A molecular marker, detection primer and application of fast-growing trait of Litopenaeus vannamei

Abstract

The invention discloses a molecular marker, detection primer and application of a fast-growing trait of Litopenaeus vannamei. The SNP molecular marker significantly associated with the fast-growing trait is located at the 308bp site of the nucleotide sequence shown in SEQ ID NO.1 (i.e., the P2638 site inside the DAN4-like cell wall protein encoding gene dan4), and the genotype of the site includes G / G, A / A, and G / A types. The prawn individuals with the A / A homozygous genotype of the SNP site have a significantly fast growth rate, the prawn individuals with the G / G homozygous genotype of the SNP site have a relatively slow growth rate, and the prawn individuals with the G / A heterozygous genotype of the SNP site have a medium growth rate. The SNP molecular marker and the molecular marker detection primer of the invention can be used for the evaluation of the characteristics of the growth trait of the population, and can also be used for the selection of prawn individuals with potential growth advantage traits.

Description

Technical Field

[0001] The invention belongs to the field of aquatic animal breeding, and in particular relates to a molecular marker, a detection primer and an application of a fast-growing trait of Litopenaeus vannamei. Background Art

[0002] The seed industry is the "chip" of agriculture, and the seed industry of Penaeus vannamei is crucial to supporting the development of the entire shrimp farming industry.

[0003] The rapid growth of shrimp can significantly reduce the cost of breeding and the emergency risks such as disease outbreaks and climate disasters caused by the long breeding cycle. Therefore, the fast growth trait of shrimp has always been the main trait in the selection and breeding of new shrimp varieties. In the past decade, 14 new varieties of white shrimp have been cultivated (as of February 2023), but the existing new varieties of Penaeus vannamei are all obtained through group or family selection technology. The application of molecular marker-assisted breeding technology in the selection and breeding of new varieties of Penaeus vannamei is still very rare.

[0004] Single nucleotide polymorphism (SNP) belongs to the third generation of molecular markers. SNP is abundant in the genome of all organisms and has a low mutation rate. With the widespread popularization of sequencing technology and the significant reduction in costs, SNP is widely used in the study of genetic diversity, phylogenetic analysis, lineage identification, genetic disease-related genes and genetic breeding. SNP molecular markers have been widely used in the breeding of terrestrial animals and plants, but their application in the field of shrimp breeding is still very limited.

[0005] Patent CN109971865A discloses a SNP marker significantly associated with the weight trait of Penaeus vannamei and its application, but does not disclose the location of the sequence where the SNP marker is located and the related genes. Growth is a complex quantitative trait controlled by multiple genes, so it is necessary to continuously explore functional genes related to shrimp growth and SNP molecular markers related to dominant growth traits. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker, detection primer and application of the fast-growing trait of Penaeus vannamei. The detection primer of the present invention can quickly identify the SNP molecular marker significantly related to the fast-growing trait of Penaeus vannamei, and quickly screen the backup parents with faster growth according to the genotype of the SNP molecular marker, which is conducive to shortening the breeding cycle of the fast-growing Penaeus vannamei strain and providing technical support for improving the growth rate of the offspring of Penaeus vannamei.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A SNP molecular marker significantly associated with the fast growth trait of Penaeus vannamei is located at the P2638 site (dan4-2638) inside the DAN4-like cell wall protein encoding gene (dan4, gene ID: LOC113802606). The SNP molecular marker was obtained through genome-wide association analysis (GWAS). The genotypes of this site include G / G, A / A, and G / A types, among which G / G and A / A are homozygous genotypes, and G / A is a heterozygous genotype.

[0009] Specifically, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, the 308 bp position of the sequence is the SNP site (corresponding to the above-mentioned dan4-2638 site), and the base R is A or G.

[0010] A detection primer for a SNP molecular marker significantly associated with the fast growth trait of Penaeus vannamei, the sequence of which is:

[0011] dan4-2638F: 5'-GGTCCAGAAGGAAGTGTAGTCT-3' (as shown in SEQ ID NO. 2);

[0012] dan4-2638R: 5'-CTCTTCCGTCTCTGGTGATGA-3' (as shown in SEQ ID NO. 3).

[0013] The PCR amplification fragment of the dan4-2638F / dan4-2638R detection primers was 549 bp in length, and the amplified fragments were all within the exons.

[0014] A kit comprising the above-mentioned detection primers dan4-2638F / dan4-2638R.

[0015] On the other hand, the present invention also provides the use of the above-mentioned SNP molecular marker, detection primer or kit in detecting the fast growth trait of Penaeus vannamei.

[0016] The SNP molecular marker is located at the 308bp site of the nucleotide sequence shown in SEQ ID NO.1, and the genotypes of the SNP site are A / A homozygous, G / G homozygous and G / A heterozygous; the shrimp individuals with the A / A homozygous genotype of the SNP site have a significantly fast growth rate, the shrimp individuals with the G / G homozygous genotype of the SNP site have a relatively slow growth rate, and the shrimp individuals with the G / A heterozygous genotype of the SNP site have a medium growth rate.

[0017] On the other hand, the present invention also provides a method for breeding fast-growing varieties of Penaeus vannamei based on the SNP molecular marker and detection primers, comprising the following steps:

[0018] (1) extracting genomic DNA of Penaeus vannamei to be tested;

[0019] (2) using the detection primers dan4-2638F / dan4-2638R to perform PCR amplification on the genomic DNA of the tested Litopenaeus vannamei;

[0020] (3) sequencing the PCR amplification products and typing the SNP molecular markers related to the fast growth trait of Penaeus vannamei according to the sequencing peak graph;

[0021] (4) Select shrimp individuals with homozygous genotype A / A as parents and mate them to produce the next generation breeding population.

[0022] The specific steps are:

[0023] The genomic DNA from shrimp tissues was extracted using a marine animal genome extraction kit (Tiangen Biochemical, China), and the extracted genomic DNA was eluted in 50 μL ddH2O and stored at -20°C.

[0024] A conventional PCR amplification system (50 μL) was prepared, including: 1 μL of shrimp genomic DNA template, 25 μL of 2× PrimeSTAR high-fidelity DNA polymerase (TaKaRa, China), 1 μL of upstream primer dan4-2638F (10 μM), 1 μL of downstream primer dan4-2638R (10 μM), and 22 μL of ddH2O.

[0025] PCR product amplification program: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 56°C for 15 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 5 min.

[0026] The PCR amplification products were submitted to a sequencing company (Shanghai Bioengineering, China) for routine PCR product sequencing, and the sequencing files were opened with BioEdit software. If the sequencing result of the dan4-2638 site was a single peak A, it was an A / A homozygous genotype, if the sequencing result of the dan4-2638 site was a single peak G, it was a G / G homozygous genotype, and if the sequencing result of the dan4-2638 site was a double peak, it was a G / A heterozygous genotype.

[0027] The shrimp individuals with A / A homozygous genotype at dan4-2638 locus had a significantly faster growth rate, the shrimp individuals with G / G homozygous genotype at dan4-2638 locus had a relatively slow growth rate, and the shrimp individuals with G / A heterozygous genotype at dan4-2638 locus had a medium growth rate.

[0028] On the other hand, the present invention also provides the use of the above-mentioned SNP molecular marker, detection primer or kit in the breeding of Penaeus vannamei.

[0029] The SNP molecular markers and molecular marker detection primers of the present invention can be used to evaluate the growth trait characteristics of a population. The SNP molecular markers and molecular marker detection primers of the present invention can also be used to select shrimp individuals with potential growth advantage traits and directly used for parent cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the electrophoresis of the PCR amplification product of the dan4-2638 site in the genome of Penaeus vannamei. M: DNAMarker DL 2000; 1-4: electrophoresis results of the amplification product of the dan4-2638 site of 4 shrimps; C: ddH2O was used as the negative control template.

[0031] Figure 2 The results of genotype identification of the dan4-2638 locus in the genome of Penaeus vannamei (sequence reverse complement). A: A / A homozygous genotype; B: G / G homozygous genotype; C: G / A heterozygous genotype. DETAILED DESCRIPTION

[0032] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0033] Example 1

[0034] Obtaining SNP molecular markers significantly associated with the fast growth trait of Litopenaeus vannamei:

[0035] (1) Sampling and trait grading of Penaeus vannamei

[0036] Shrimp samples were collected from different farms and markets. If the size difference of the same batch of shrimp was obvious, it was used as a batch for sampling. 50 shrimps were taken from each batch, and the growth rate trait (GW) was characterized by body length. The shrimps in each batch were divided into four grades according to body length: fast, fast, normal, and slow. A total of 75 shrimps with growth difference traits characterized by body length were collected. In addition, 75 samples were collected for Vibrio infection traits (AB), 75 samples were collected for Enterocytozoa infection traits (EH), and 75 samples were collected for hepatopancreas index (EI), totaling 300 samples.

[0037] (2) Whole-genome association analysis to discover SNPs associated with fast growth traits

[0038] DNA was extracted from 300 samples, and conventional second-generation sequencing libraries were constructed and sequenced (Beijing Biomarker Biotechnology Co., Ltd.). The sequencing results were compared with the reference genome of Penaeus vannamei. GATK (V3.4.6) software was used to search for SNPs and InDels. SNP sites were filtered based on minor allele frequency and site integrity, and high consistency and confirmed SNP sites were retained. Combined with EMMAX software, a mixed linear model was used for association analysis, and Bonrroni correction was used for candidate sites in trait association analysis (Beijing Biomarker Biotechnology Co., Ltd.).

[0039] (3) Mining of molecular markers associated with fast growth traits

[0040] According to the P value of the SNP sites associated with the traits analyzed by GWAS, they were arranged and unique SNP sites significantly associated with the fast growth trait were screened. Then, SNPs located in the promoter region of the gene and SNPs that caused amino acid coding mutations within the gene were screened. The results showed that the P2638 site (dan4-2638) within the DAN4-like cell wall protein encoding gene (dan4, gene ID: LOC113802606) was significantly associated with the fast growth trait of shrimp (P<0.05). The GWAS analysis results showed that shrimp individuals with the A / A homozygous genotype at the dan4-2638 site had a faster growth rate, shrimp individuals with the G / G homozygous gene at the dan4-2638 site had a relatively slower growth rate, and shrimp individuals with the G / A heterozygous genotype at the dan4-2638 site had a medium growth rate.

[0041] Example 2

[0042] Primer design for detecting genotype of dan4-2638 locus related to fast growth trait of Litopenaeus vannamei:

[0043] Based on the sequence of dan4 gene (LOC113802606), Primer 6.0 was used to design PCR amplification primers for dan4-2638 locus. The primer design requirements were as follows:

[0044] 1) The dan4-2638 site is located near the middle of the designed amplification sequence to avoid amplification and sequencing errors;

[0045] 2) The length of the amplified fragment is between 400-600 bp;

[0046] 3) The anchor sites of the amplification primers are all located in the exon region;

[0047] 4) The amplified product has a single band and strong specificity.

[0048] Through primer design and PCR amplification testing, it was finally found that dan4-2638F / dan4-2638R can obtain the best PCR amplification results. The sequence of the dan4-2638F / dan4-2638R primer combination is as follows:

[0049] dan4-2638F: 5'-GGTCCAGAAGGAAGTGTAGTCT-3' (as shown in SEQ ID NO. 2);

[0050] dan4-2638R: 5'-CTCTTCCGTCTCTGGTGATGA-3' (as shown in SEQ ID NO. 3).

[0051] The length of the PCR amplified fragment of the dan4-2638F / dan4-2638R primer combination is 549 bp (the sequence is shown in SEQ ID NO.1), and the amplified fragment is all within the exon.

[0052] >From 2331 to 2879 (sequence shown in SEQ ID NO.1)

[0053] .

[0054] The SNP molecular marker associated with the fast growth trait of Penaeus vannamei is located at the 308bp position (i.e., dan4-2638 position) of the nucleotide sequence shown in SEQ ID NO.1, the base R at this position is A or G, and the genotype is G / G, A / A or G / A.

[0055] Example 3

[0056] PCR and sequencing detection of the genotype of the fast-growing trait of Penaeus vannamei at the dan4-2638 locus:

[0057] The detection of molecular marker locus genes using the above dan4-2638F / dan4-2638R primer combination comprises the following steps:

[0058] (1) Shrimp genome extraction

[0059] The ends of the shrimp appendages were cut or feces samples were taken, and the shrimp genomic DNA was extracted using the Marine Animal Genome Extraction Kit (Tiangen Biochemical, China).

[0060] (2) Preparation of PCR amplification system

[0061] A conventional PCR amplification system (50 μL) was prepared, including: 1 μL of shrimp genomic DNA template, 25 μL of 2× PrimeSTAR high-fidelity DNA polymerase (TaKaRa, China), 1 μL of upstream primer dan4-2638F (10 μM), 1 μL of downstream primer dan4-2638R (10 μM), and 22 μL of ddH2O.

[0062] (3) PCR amplification

[0063] PCR product amplification program: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 56°C for 15 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 5 min.

[0064] (4) PCR product sequencing and result interpretation

[0065] The electrophoresis of the PCR amplification product of the region where the dan4-2638 site of the vannamei genome is located is shown in Figure 1 shown.

[0066] The PCR amplification products were submitted to a sequencing company (Shanghai Bioengineering, China) for routine PCR product sequencing, and the sequencing files were opened with BioEdit software. If the sequencing result of the dan4-2638 site is a single peak A, it is an A / A homozygous genotype, if the sequencing result of the dan4-2638 site is a single peak G, it is a G / G homozygous genotype, and if the sequencing result of the dan4-2638 site is a double peak, it is a G / A heterozygous genotype. Figure 2 shown.

[0067] Example 4

[0068] Verification of molecular markers at dan4-2638 locus related to fast growth traits of Litopenaeus vannamei:

[0069] 75 shrimps of the same batch with obvious size differences were collected. The weight phenotype data of the 75 shrimps are shown in Table 1. According to the statistical results, the mean weight of all samples was (15.76±3.52) g, the coefficient of variation was 22.34%, the maximum weight was 23.44 g, and the minimum weight was 8.90 g.

[0070] The growth rate was characterized by body weight, and the 75 shrimps were divided into three growth rate levels according to their body weight: fast growth (W ≥ 17g), normal growth (17g > W ≥ 14g), and slow growth (14g > W ≥ 8g). According to Table 1, the average body weights of the three growth rate levels of fast growth, normal growth, and slow growth were 19.27g, 15.58g, and 11.17g, respectively. It can be seen that the growth traits of the three growth rate shrimp samples are significantly different.

[0071] Table 1 Statistical data of body weight of 75 Penaeus vannamei with different growth rate traits (n=75)

[0072]

[0073]

[0074] The genotype of dan4-2638 locus of 75 shrimps with size difference traits were detected using the detection primers dan4-2638F / dan4-2638R of Example 2 and the genotype detection method of Example 3. The detection results are shown in Table 2:

[0075] Table 2 Genotype statistics of dan4-2638 locus of 75 Penaeus vannamei with size difference traits (n=75)

[0076]

[0077]

[0078] *Bold: Shrimp classified as normal growth rate have A / A homozygous genotype; Underline: Shrimp classified as fast growth have G / A heterozygous genotype; Italic bold: Shrimp classified as fast growth have G / G heterozygous genotype.

[0079] As shown in Table 2, the average weight of shrimps with A / A genotype at dan4-2638 was 19.23g, the average weight of shrimps with G / A genotype at dan4-2638 was 15.97g, and the average weight of shrimps with G / G genotype at dan4-2638 was 11.90g. The average weight of shrimps with A / A genotype was 1.62 times that of shrimps with G / G genotype. Among the 30 shrimps with fast-growing phenotypic traits, 26 had A / A genotype at dan4-2638, accounting for 86.7%. Among them, the weight of the 3 shrimps with A / A genotype that were originally classified as medium growth rate (thickness in Table 2) was also very close to the threshold of the fast-growing individual classification standard (W≥17g). These results show that the use of A / A genotype at dan4-2638 to determine whether Vannamei shrimp has the potential for fast growth traits has a high accuracy. The shrimp individuals with A / A homozygous genotype at dan4-2638 locus had a significantly faster growth rate, the shrimp individuals with G / G homozygous genotype at dan4-2638 locus had a relatively slow growth rate, and the shrimp individuals with G / A heterozygous genotype at dan4-2638 locus had a medium growth rate.

[0080] Example 5

[0081] Application of molecular markers and detection primers for the dan4-2638 locus associated with the fast growth trait of Penaeus vannamei:

[0082] Vannamei shrimp populations G1, G2, and G3 from three different sources were collected, 50 tails were randomly sampled, and the shrimp genome was extracted, PCR amplified, and sequenced using the method in Example 3. The test results showed that the proportions of the A / A genotype at the dan4-2638 site of the vannamei shrimp populations G1, G2, and G3 were 28%, 16%, and 0%, respectively. Therefore, the vannamei shrimp population G1 can be used as a candidate population for fast growth.

[0083] The vannamei shrimp population G1 was cultivated to adult shrimp (≥20 g), and large shrimp individuals were selected from them. Fecal samples were collected tail by tail, and the shrimp genome was extracted, PCR amplified and sequenced using the method in Example 3. The shrimp individuals with A / A genotype at the dan4-2638 site were retained and continued to be cultivated into parents with fast growth traits.

[0084] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of SNP molecular markers significantly associated with the fast growth trait of Penaeus vannamei in detecting the fast growth trait of Penaeus vannamei, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, the 308bp position of the sequence is a SNP site, and the base R is A or G; the shrimp individuals with the A / A homozygous genotype of the SNP site have a significantly fast growth rate, the shrimp individuals with the G / G homozygous genotype of the SNP site have a relatively slow growth rate, and the shrimp individuals with the G / A heterozygous genotype of the SNP site have a medium growth rate.

2. A method for breeding a fast-growing variety of Penaeus vannamei, characterized in that: The following steps are involved: (1) Extracting genomic DNA of Penaeus vannamei to be tested; (2) PCR amplification of the genomic DNA of Penaeus vannamei was performed using the detection primers dan4-2638F: 5'-GGTCCAGAAGGAAGTGTAGTCT-3' and dan4-2638R: 5'-CTCTTCCGTCTCTGGTGATGA-3'; (3) sequencing the PCR amplification product, and typing the SNP molecular markers related to the fast growth trait of the vannamei shrimp in claim 1 according to the sequencing peak graph; (4) Select shrimp individuals with homozygous genotype A / A as parents and mate them to produce the next generation breeding population.

3. The method according to claim 2, characterized in that The PCR amplification system is: 50 μL, including 1 μL of shrimp genomic DNA template, 25 μL of 2×PrimeSTAR high-fidelity DNA polymerase, 1 μL of 10 μM upstream primer dan4-2638F, 1 μL of 10 μM downstream primer dan4-2638R, and 22 μL of ddH2O.

4. The method according to claim 2, characterized in that: The PCR amplification program was as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 56°C for 15 sec, extension at 72°C for 30 sec, 30 cycles; and extension at 72°C for 5 min.

5. Application of SNP molecular markers significantly associated with the fast growth trait of Penaeus vannamei in breeding of Penaeus vannamei, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, the 308bp position of the sequence is a SNP site, and the base R is A or G; the shrimp individuals with the A / A homozygous genotype of the SNP site have a significantly fast growth rate, the shrimp individuals with the G / G homozygous genotype of the SNP site have a relatively slow growth rate, and the shrimp individuals with the G / A heterozygous genotype of the SNP site have a medium growth rate.

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