A Litopenaeus vannamei PDE11A gene, SNP molecular marker combination, detection primer and its application

Abstract

The present invention discloses a Litopenaeus vannamei PDE11A gene, SNP molecular marker combination, detection primer and their applications. The nucleotide sequence of the Litopenaeus vannamei PDE11A gene is SEQ ID NO.1. The SNP molecular marker combination is located on the SEQ ID NO.1 sequence and includes a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2, with a polymorphism of A / G; the second SNP molecular marker is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3, with a polymorphism of A / G. Selecting parents with the AA / AA genotype in the SNP molecular marker combination can improve the growth rate of the breeding population, provide an innovative tool for the molecular breeding of Litopenaeus vannamei, shorten the breeding cycle, and contribute to the development of the industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and particularly relates to a combination of Litopenaeus vannamei PDE11A gene, SNP molecular markers, detection primers and their applications. Background Art

[0002] Litopenaeus vannamei, also known as whiteleg shrimp or Pacific white shrimp, belongs to the phylum Arthropoda, class Crustacea, order Decapoda, family Penaeidae, genus Penaeus. It is a pillar species in the global aquaculture industry, with the highest aquaculture production and one of the highest single-output aquaculture species. Litopenaeus vannamei is widely cultured due to its fast growth rate, strong environmental adaptability and high economic value. However, during the aquaculture process, there are significant differences in the growth performance of Litopenaeus vannamei. The growth traits of shrimp are mainly affected by various genetic and environmental factors. Among them, body weight and growth rate are important indicators to measure its economic value. Light body weight or slow growth rate will seriously affect the aquaculture efficiency and economic benefits.

[0003] The molecular breeding of Litopenaeus vannamei mainly relies on traditional breeding selection methods. Although these methods can improve growth traits to a certain extent, they have the following defects: (1) Long breeding cycle and low efficiency: Traditional breeding methods select better-performing individuals for reproduction. However, due to the strong quantitative and environmental influence of growth traits, the breeding cycle is long and the efficiency is low, making it difficult to meet the growing market demand; (2) Lack of precise molecular markers: Although some genomics technologies (such as genomic selection, marker-assisted selection) have been applied to the breeding of shrimp, there is still a lack of efficient molecular markers for growth traits. Existing markers are often more general and cannot accurately identify genetic factors highly related to growth traits, resulting in limited efficiency of genetic improvement; (3) Difficulty in achieving the precision of genetic improvement: Due to the complex genetic basis of the growth traits of Litopenaeus vannamei, traditional phenotypic selection methods are often greatly affected by environmental factors, making it difficult to achieve precise screening and transmission of excellent growth traits.

[0004] In recent years, the rapid development of molecular breeding technology has brought new ideas to the aquaculture industry. SNP (Single Nucleotide Polymorphism) refers to the polymorphism of single nucleotides in the genome. As the third-generation molecular marker, SNP molecular marker is the most widely used and latest molecular marker at present, with the characteristics of high density, strong stability, and co-dominance. It is the most widely used molecular marker technology in economic crustaceans such as shrimp and crab. The growth traits of Litopenaeus vannamei, as a complex quantitative trait, still need to further explore and verify new genetic markers.

[0005] Phosphodiesterase 11A gene (PDE11A, Phosphodiesterase 11A) belongs to a member of the phosphodiesterase (PDE) superfamily. This gene encodes a bifunctional 3',5'-cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterase, which can catalyze the degradation of cAMP and cGMP into the corresponding 5'-monophosphates, thus playing an important role in cell signal transduction. By regulating the cAMP and cGMP signaling pathways, PDE11A has potential functional regulatory effects in a variety of biological processes. In Litopenaeus vannamei, the PDE11A gene (on chromosome 37, LOC113813671 (updated on December 12, 2024); derived from the genomic sequence NW_020869897.1: 217033-239857 segment, a total of 223825 bp; website: Record suppressed: Penaeus vannamei breed Kehai No.1 unplaced genomic scaffold, ASM378908 - Nucleotide - NCBI ) has not been reported, and there is no research to explore the relationship between this gene and the growth traits of shrimp.

[0006] Therefore, developing SNP molecular markers on the PDE11A gene of Litopenaeus vannamei growth traits is of great significance for the breeding of new varieties of Litopenaeus vannamei, providing innovative tools for the molecular breeding of Litopenaeus vannamei, shortening the breeding cycle, and boosting industrial development and efficiency improvement. Summary of the Invention

[0007] The object of the present invention is to provide a PDE11A gene of Litopenaeus vannamei growth traits, a combination of SNP molecular markers, detection primers, and apply them to the breeding of fast-growing varieties of shrimp, accelerate the genetic improvement of growth traits, improve breeding efficiency and economic benefits. To achieve the above object:

[0008] On the one hand, the present invention provides a PDE11A gene of Litopenaeus vannamei, and the nucleotide sequence of the PDE11A gene of Litopenaeus vannamei is shown as SEQ ID NO.1.

[0009] On the other hand, the present invention provides the application of the PDE11A gene of Litopenaeus vannamei in the breeding of Litopenaeus vannamei.

[0010] On the other hand, the present invention provides a SNP molecular marker combination, and the sequence where the SNP molecular marker combination is located is as shown in SEQ ID NO.1; the SNP molecular marker combination includes a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2; the polymorphism of the first SNP molecular marker is A / G; the second SNP molecular marker is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3; the polymorphism of the second SNP molecular marker is A / G.

[0011] On the other hand, the present invention provides an application of a SNP molecular marker combination in the breeding of Litopenaeus vannamei.

[0012] Furthermore, the breeding of Litopenaeus vannamei is for the body length screening of Litopenaeus vannamei, the identification of broodstock individuals, population breeding or strain maintenance.

[0013] On the other hand, the present invention provides a primer set, which contains one, a pair or multiple of the following base sequences: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3';

[0014] PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3';

[0015] PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3';

[0016] PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3';

[0017] Furthermore, the primer set is used to detect the SNP molecular marker combination, and the sequence where the SNP molecular marker combination is located is as shown in SEQ ID NO.1; the SNP molecular marker combination includes a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2; the polymorphism of the first SNP molecular marker is A / G, and PDE11A-2877-Fw and PDE11A-2877-Rw are used to detect the first SNP molecular marker; the second SNP molecular marker is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3; the polymorphism of the second SNP molecular marker is A / G, and PDE11A-8932-Fw and PDE11A-8932-Rw are used to detect the second SNP molecular marker.

[0018] On the other hand, the present invention provides an application of a primer set in the breeding of Litopenaeus vannamei.

[0019] On the other hand, the present invention provides a detection kit comprising a primer set.

[0020] On the other hand, the present invention provides an application of a detection kit in the breeding of Litopenaeus vannamei.

[0021] On the other hand, the present invention provides a method for breeding Litopenaeus vannamei using a SNP molecular marker combination, comprising the following steps: Step A: Detect the SNP molecular marker combination of Litopenaeus vannamei to obtain the genotype of the SNP combination site; Step B: Screen according to the genotype of the SNP combination site; the genotypes of the SNP combination sites are AA / AA, AA / AG, AA / GG, AG / AG, AG / GG or GG / GG genotypes; the screening basis is the genotype-body length rule; Step C: Breed Litopenaeus vannamei with the genotype of AA / AA at the SNP combination site.

[0022] Further, Step A includes: Step A-1: Extract the genomic DNA of Litopenaeus vannamei; Step A-2: Using the genomic DNA of Litopenaeus vannamei as a template, perform PCR amplification using the following primer pairs:

[0023] PDE11A-2877-Fw: 5’-AGTGACTCGTAGCGAAAGCC-3’;

[0024] PDE11A-2877-Rw: 5’-CCTGTGTATGCCACGCTCTT-3’;

[0025] PDE11A-8932-Fw: 5’-TGCCTTGAAATGACGTGTGTG-3’;

[0026] PDE11A-8932-Rw: 5’-ACCACTGGGTAGTCTGAAATG-3’;

[0027] The ratio of the components in the PCR amplification system is as follows: 1 - 2 μL of Litopenaeus vannamei genomic DNA (100 ng / μL); 1 - 2 μL of forward primer Fw (10 μM); 1 - 2 μL of reverse primer Rw (10 μM); 10 - 13 μL of 2×Taq Plus Master Mix Ⅱ (Plus); 8 - 10 μL of enzyme - free and sterile water. The PCR amplification procedure successively includes: pre - denaturation at 95°C for 3 min; then 32 cycles: 95°C for 15 s; 56 - 58°C for 20 - 30 s; 72°C for 40 s; final extension at 72°C for 10 min to obtain the PCR amplification product. Step A - 3: Sequencing is performed using the PCR amplification product to obtain the genotypes of the SNP combination sites. The genotypes of the SNP combination sites are A / G at the 2877th position and A / G at the 8932nd position of the sequence shown in SEQ ID NO.1. A / G indicates that there is A / G polymorphism at this SNP site, and the A genotype is superior to the G genotype.

[0028] Furthermore, the first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2; the polymorphism of the first SNP molecular marker is A / G. The second SNP molecular marker is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3; the polymorphism of the second SNP molecular marker is A / G.

[0029] Furthermore, the screening criterion is the body length of Litopenaeus vannamei: The body length of Litopenaeus vannamei with the AA / AA genotype at the SNP combination site > The body length of Litopenaeus vannamei with the AA / AG genotype at the SNP combination site > The body length of Litopenaeus vannamei with the AA / GG genotype at the SNP combination site > The body length of Litopenaeus vannamei with the AG / AG genotype at the SNP combination site > The body length of Litopenaeus vannamei with the AG / GG genotype at the SNP combination site > The body length of Litopenaeus vannamei with the GG / GG genotype at the SNP combination site.

[0030] The present invention locates two SNP molecular marker sites related to body length on the PDE11A gene of Litopenaeus vannamei by using genome-wide association analysis, which are SNP molecular marker combinations, named PDE11A-2877 (the first SNP site) and PDE11A-8932 (the second SNP site) respectively. Among them, the sequence of the PDE11A-2877 SNP molecular marker is shown in SEQ ID NO.2, the SNP site is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2, and the polymorphism is A / G type; the sequence of the PDE11A-8932 SNP molecular marker is shown in SEQ ID NO.3, the SNP site is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3, and the polymorphism is A / G type; the primers SEQ ID NO.4 and SEQ ID NO.5 designed according to the SEQ ID NO.2 sequence can detect the PDE11A-2877 SNP molecular marker, and the primers SEQ ID NO.6 and SEQ ID NO.7 designed according to the SEQ ID NO.3 sequence can detect the PDE11A-8932 SNP molecular marker, and the above primers can also be prepared into a kit. Therefore, the two SNP molecular markers PDE11A-2877 and PDE11A-8932 provided by the present invention can be used to assist in the selection of fast-growing parents. By selecting parents with the AA / AA genotype in this SNP molecular marker combination, the growth rate of the breeding population can be increased, providing an innovative tool for the molecular breeding of Litopenaeus vannamei, shortening the breeding cycle, and contributing to the development of the industry and the improvement of efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above content of the present invention and the following specific embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are only examples of the claimed technical solutions.

[0032] Figure 1 It is the Manhattan plot of genome-wide association analysis of the body length of Litopenaeus vannamei in Example 1;

[0033] Figure 2 It is the agarose gel electrophoresis pattern of the genomic DNA of Litopenaeus vannamei in Example 1 (where M is Marker; A is the genomic DNA of Litopenaeus vannamei numbered 1-12; B is the genomic DNA of Litopenaeus vannamei numbered 13-24, and the length exceeds 5000 bp and the integrity is good);

[0034] Figure 3Agarose gel electrophoresis diagram of the PCR amplification products of SEQ ID NO.2 and SEQ ID NO.3 of Litopenaeus vannamei in Example 1 (where M is Marker; in a, 1-12 are the PCR products of the PDE11A-2877 locus, with a relatively bright band at around 441 bp; in b, 1-12 are the PCR products of the PDE11A-8932 locus, with a relatively bright band at around 692 bp);

[0035] Figure 4 Diagram for the sequencing process description in Example 1;

[0036] Figure 5 Reverse sequencing peak diagram of the PCR amplification product of SEQ ID NO.2 in Example 1 (SEQ ID NO.10 has polymorphism at the 289th position of the base);

[0037] Figure 6 Forward sequencing peak diagram of the PCR amplification product of SEQ ID NO.3 in Example 1 (SEQ ID NO.11 has polymorphism at the 154th position of the base);

[0038] Figure 7 Length distribution of different genotype individuals of Litopenaeus vannamei in Example 1 (where the abscissa represents the genotype, in sequence AA / AA, AA / AG, AA / GG, AG / AG, AG / GG, and GG / GG; the ordinate is the body length of Litopenaeus vannamei, with the unit of (mm); "***" represents that there is a significant difference in the body length of Litopenaeus vannamei individuals between two genotypes);

[0039] Figure 8 Diagram of the body length growth of Litopenaeus vannamei after RNA interference in Example 2 (where the abscissa is the body length growth, with the unit of (cm); the ordinate is different RNA interference situations, green is the blank control group (PBS), purple is the negative control group (siNC), and orange is the experimental group (siPDE11A));

[0040] Figure 9 Agarose gel electrophoresis diagram of the total RNA of the hepatopancreas and muscle of Litopenaeus vannamei after RNA interference (where M is Marker, in a, 1-12 are the total RNA of the hepatopancreas after RNA interference, with a relatively bright band at around 500 bp; in b, 1-12 are the total RNA of the muscle after RNA interference, with a relatively bright band at around 500 bp);

[0041] Figure 10Relative expression levels of the Litopenaeus vannamei PDE11A gene in the hepatopancreas and muscle after RNA interference (where the abscissa represents the hepatopancreas and muscle; the ordinate represents the relative expression level; green is the blank control group (PBS), purple is the negative control group (siNC), orange is the experimental group (siPDE11A); "***" represents that there is a significant difference in the body length of Litopenaeus vannamei individuals between the two genotypes). Detailed implementation manners

[0042] The detailed features and advantages of the present invention are described in detail in the following detailed implementation manners. The content is sufficient for any person skilled in the art to understand the technical content of the present invention and implement it accordingly. According to the specification, claims and drawings disclosed in this specification, those skilled in the art can easily understand the related purposes and advantages of the present invention.

[0043] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0044] To make the objectives, technical solutions and advantages of the present invention clearer, the implementation manners of the present invention will be further described in detail below with reference to the drawings. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0045] (1) Source of sample materials

[0046] In this implementation manner, Litopenaeus vannamei was purchased from Hainan Renhai Aquatic Science and Technology Co., Ltd. (Wenchang City, Hainan Province).

[0047] (2) Source of reagents and consumables

[0048] Table 1 Sources and catalog numbers of reagents and consumables required for the experiment

[0049] ;

[0050] (3) Source of instruments and equipment

[0051] Table 2 Sources and models of instruments and equipment required for the experiment

[0052] ;

[0053] Example 1

[0054] A SNP molecular marker combination associated with the body length growth trait of Litopenaeus vannamei, comprising the following steps:

[0055] S1. Sample collection and sequencing, specifically referring to the following steps:

[0056] (1) A total of 996 individuals were randomly selected from an 8-month-old Litopenaeus vannamei population. The body length of each Litopenaeus vannamei was measured with a ruler (accurate to 1 mm), and the shrimp antenna samples were collected for genomic analysis. The shrimp antenna samples of Litopenaeus vannamei were sent to Novogene Co., Ltd. in Beijing for genome resequencing. A total of 14,276,173 SNP loci were obtained. After quality control of the above 14,276,173 SNP loci, a total of 2,706,353 SNP loci were screened out.

[0057] (2) According to the gene resequencing results in step (1) above, using the genome-wide SNP genotyping data of the 2,706,353 SNP loci that have been screened out, the principal component analysis (PCA) and population structure analysis were performed using the PLINK software. The ten principal components, population structure matrix, and gender were used as covariates to control the interference of population structure and gender on the analysis results. The compressed mixed linear model (CMLM) of the GAPIT package was used to combine the SNP genotypes and phenotypic data of Litopenaeus vannamei for genome-wide association study (GWAS, Genome-Wide Association Study).

[0058] The formula is as follows:

[0059] ;

[0060] where Y is an n×1 phenotypic vector containing the phenotypic values of n individuals. X is an n×p design matrix, including independent variables of fixed effects, such as SNP genotypes and other covariates (gender, age, etc.). β is a p×1 vector of fixed effect coefficients, representing the fixed effect of SNP genotypes on the phenotype. Zc is an n×q compressed design matrix used to represent random effects. uc is a q×1 random effect vector, usually assumed to follow a multivariate normal distribution uc~N(0, Iq), where Iq is a q×q identity matrix, is the variance of the random effect. is an n×1 error vector, assuming ~N(0, In), where In is an n×n identity matrix, is the variance of the error.

[0061] According to the Bonferroni correction α Bonferroni = α / m, where α is the global significance level (usually taken as 0.05), and m is the number of independent tests performed (usually the number of SNPs tested).

[0062] The results are as Figure 1As shown, the significance threshold for determining the degree of association between SNPs and the body length trait of Litopenaeus vannamei through GWAS analysis is 1.85×10 -8 , and the SNPs associated through GWAS analysis are sorted according to the P-value size, and unique SNP loci significantly related to the body length growth trait of Litopenaeus vannamei are screened out. The results show that there are two SNP molecular marker loci on the PDE11A gene (LOC113813671; SEQ ID NO.1) of Litopenaeus vannamei, which are SNP molecular marker combinations, named PDE11A-2877 (the first SNP locus) and PDE11A-8932 (the second SNP locus), respectively. Among them, PDE11A-2877 is located at the 2877th position of SEQ ID NO.1 in the PDE11A gene on chromosome 37 of Litopenaeus vannamei. The nucleotide sequence where PDE11A-2877 is located is as shown in SEQ ID NO.2. Through genotyping, the genotypes of PDE11A-2877 are AA, AG, and GG. As shown in SEQ ID NO.2, the 14th base from the 5' end is A or G, which is an A / G type SNP molecular marker. PDE11A-8932 is located at the 8932nd position of SEQ ID NO.1 in the PDE11A gene on chromosome 37 of Litopenaeus vannamei. The nucleotide sequence where PDE11A-8932 is located is as shown in SEQ ID NO.3. Through genotyping, the genotypes of PDE11A-8932 are AA, AG, and GG. As shown in SEQ ID NO.3, the 217th base from the 5' end is A or G, which is an A / G type SNP molecular marker.

[0063] S2. Extraction of genomic DNA from Litopenaeus vannamei

[0064] To further verify the genotypes with obvious advantages, 200 large and small shrimps were randomly selected from a 9-month-old Litopenaeus vannamei population. The shrimp antennae of Litopenaeus vannamei were cut and ground into powder using liquid nitrogen. The genomic DNA of Litopenaeus vannamei was extracted using a marine animal genomic DNA extraction kit (Tiangen Biochemical, and all the following reagents and consumables are from this kit). The DNA extraction steps are as follows:

[0065] (1) Sample pretreatment: Weigh 30 mg of shrimp antennae, add 200 μL of GA buffer to a centrifuge tube, mix gently, and shake for 15 seconds to ensure that the shrimp antennae tissue is completely wetted.

[0066] (2) Proteinase K digestion: Add 20 μL of Proteinase K solution (20 mg / mL) to the centrifuge tube, mix well, and incubate in a 56°C incubator until the tissue is completely dissolved.

[0067] (3) Lysis and precipitation: Add 200 μL of GB lysis buffer, mix well, and place in a 70°C water bath for 10 minutes. Then add 200 μL of absolute ethanol and mix well.

[0068] (4) Adsorption column purification: Transfer the mixture to a CB3 adsorption column, place it in a collection tube, centrifuge at 12,000 rpm for 30 seconds, and then discard the waste liquid. Then add 500 μL of GD buffer and 600 μL of PW wash buffer respectively, centrifuge for 30 seconds each time to wash the adsorption column to remove impurities.

[0069] (5) Adsorption column drying: Place the washed adsorption column in a centrifuge tube again, centrifuge at 12,000 rpm for 2 minutes, and then let it stand at room temperature for 2 - 5 minutes to ensure that the ethanol in the adsorption column is completely evaporated.

[0070] (6) DNA elution: Place the adsorption column in a new centrifuge tube, add 50 - 200 μL of sterilized water to the middle of the adsorption column, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 2 minutes to collect the eluted DNA solution.

[0071] (7) Quality detection and preservation: Use a spectrophotometer to detect the concentration and purity of DNA, ensure that OD 260 / OD 280 is between 1.8 - 2.0, and OD 260 / OD 230 is greater than 2.0.

[0072] The final concentration of the Litopenaeus vannamei genomic DNA obtained is 100 ng / μL. Use 1.5% agarose gel electrophoresis to detect the Litopenaeus vannamei genomic DNA (randomly select 24 Litopenaeus vannamei genomic DNAs for detection), and store the remaining Litopenaeus vannamei genomic DNA in a -20°C refrigerator for future use.

[0073] The results of agarose gel electrophoresis of Litopenaeus vannamei genomic DNA are as Figure 2 shown. Among them, M is the Marker, and 1 - 24 are all the target genes (Litopenaeus vannamei genomic DNA), and the length exceeds 5000 bp and the integrity is good.

[0074] S3. Primer design

[0075] Use the Primer-BLAST function module of NCBI to design amplification primers for the PDE11A - 2877 site and PDE11A - 8932 site of Litopenaeus vannamei:

[0076] PDE11A - 2877 - Fw: 5’-AGTGACTCGTAGCGAAAGCC-3’ (SEQ ID NO.4)

[0077] PDE11A-2877-Rw: 5’-CCTGTGTATGCCACGCTCTT-3’ (SEQ ID NO.5)

[0078] PDE11A-8932-Fw: 5’-TGCCTTGAAATGACGTGTGTG-3’ (SEQ ID NO.6)

[0079] PDE11A-8932-Rw: 5’-ACCACTGGGTAGTCTGAAATG-3’ (SEQ ID NO.7)

[0080] The above 4 synthesized primer sequences (SEQ ID NO.4 - 7) were sent to Beijing Tsingke Biotechnology Co., Ltd. for primer synthesis and used for the amplification of SEQ ID NO.2 and SEQ ID NO.3 sequences. Among them, SEQ ID NO.4 - 5 were used for the amplification of SEQ ID NO.2 sequence, and SEQ ID NO.6 - 7 were used for the amplification of SEQ ID NO.3 sequence.

[0081] S4, PCR Amplification Reaction

[0082] Using the Litopenaeus vannamei genomic DNA obtained in step S2 as a template, PCR amplification reactions were respectively carried out using the PDE11A-2877 and PDE11A-8932 primers synthesized in step S3. At the same time, the synthesized PDE11A-2877 and PDE11A-8932 primers were put into a kit to make a detection kit, and PCR amplification reactions could also be carried out. The specific steps are as follows:

[0083] (1) The PCR amplification reaction system (25 μL system) was designed as follows:

[0084] Litopenaeus vannamei genomic DNA (100 ng / μL), 1 μL;

[0085] Forward primer Fw (10 μM), 1 μL;

[0086] Reverse primer Rw (10 μM), 1 μL;

[0087] 2×Taq Plus Master Mix Ⅱ (Plus), 12.5 μL;

[0088] Enzyme-free and sterile water, 9.5 μL.

[0089] (2) The PCR amplification reaction program was:

[0090] Pre-denaturation at 95℃ for 3 min;

[0091] Denature at 95°C for 15 s;

[0092] Anneal at 56°C for 20 s;

[0093] Extend at 72°C for 40 s. There are a total of 32 cycles from denaturation to extension;

[0094] Fully extend at 72°C for 10 min;

[0095] Store at 4°C.

[0096] After the PCR amplification reaction, the PCR amplification products of the PDE11A-2877 site and the PCR products of the PDE11A-8932 site are obtained respectively. The above PCR amplification products are detected by 1.5% agarose gel electrophoresis.

[0097] The agarose gel electrophoresis results of the PCR amplification product of SEQ ID NO.2 are as Figure 3 shown in a. M is the Marker, 1-12 are the PCR products of the PDE11A-2877 site, and there is a relatively bright band at about 441 bp.

[0098] The agarose gel electrophoresis results of the PCR amplification product of SEQ ID NO.3 are as Figure 3 shown in b. M is the Marker, 1-12 are the PCR products of the PDE11A-8932 site, and there is a relatively bright band at about 692 bp.

[0099] Verification of S5 and SNP sites

[0100] (1) Send the PCR amplification products obtained in step S4 to Beijing Tsingke Biotechnology Co., Ltd. for sequencing analysis. The flow chart of the sequencing is as Figure 4 shown. The PCR amplification product of SEQ ID NO.2 is reverse-sequenced. The reverse sequencing primer T1 is: 5’-GTTTCAGAACACACGAGTAAAG-3’ (SEQ ID NO.8); the PCR amplification product of SEQ ID NO.3 is forward-sequenced. The forward sequencing primer T2 is: 5’-ATTCCAGAAAGTGAAGATACCC-3’ (SEQ ID NO.9), and the sequencing peak maps of the PCR amplification products are obtained ( Figure 5 , Figure 6 ).

[0101] (2) Genotype the two SNP sites according to the sequencing peak maps of the PCR amplification products obtained above ( Figure 5 , Figure 6 ).

[0102] The reverse sequencing result of the SEQ ID NO.2 sequence is shown in SEQ ID NO.10 (FX1). The sequence of SEQ ID NO.10 is a partial complementary sequence of SEQ ID NO.2, that is, the sequence length of SEQ ID NO.10 is less than the complementary sequence of SEQ ID NO.2. If there is only one T base peak at the 289th position from the 5' end in SEQ ID NO.10, the genotyping result is AA type; if there is only one C base peak at this position, the genotyping result is GG type; if there are both T base peaks and C base peaks at this position, the genotyping result is AG type.

[0103] The forward sequencing result of the SEQ ID NO.3 sequence is shown in SEQ ID NO.11 (ZX1). The sequence of SEQ ID NO.11 is a partial sequence of SEQ ID NO.3. If there is only one A base peak at the 154th position from the 5' end in SEQ ID NO.11, the genotyping result is AA type; if there is only one G base peak at this position, the genotyping result is GG type; if there are both A base peaks and G base peaks at this position, the genotyping result is AG type.

[0104] (3) One-way analysis of variance (ANOVA) was used to detect the differences in body lengths among the three genotypes AA, AG, and GG of the SNP loci in Litopenaeus vannamei. The results are shown in Table 3 and Figure 7 As shown, through the statistical analysis of the individual genotypes of Litopenaeus vannamei, it was found that the individuals with the genotype AA / AA of PDE11A-2877 and PDE11A-8932 had a significantly higher growth rate than other genotype individuals under the same breeding conditions; the individuals with the genotype GG / GG of PDE11A-2877 and PDE11A-8932 had a significantly lower growth rate than other genotype individuals under the same breeding conditions. These results prominently indicate that the genotype AA / AA of PDE11A-2877 and PDE11A-8932 has an obvious advantage in promoting the rapid growth of Litopenaeus vannamei and may be an important genetic marker affecting growth traits.

[0105] Table 3 Association between SNP molecular markers of PDE11A gene and body length of Litopenaeus vannamei

[0106] ;

[0107] Through the SNP genotyping results, individual identification of broodstock, population breeding, and strain maintenance can be carried out:

[0108] (1) Retain the male or female parent with the SNP molecular marker combination (the first SNP molecular marker and the second SNP molecular marker) both being AA type for seed retention and propagation;

[0109] (2)If both the male parent and the female parent are not of the AA genotype, select two SNP loci of the male parent and the female parent that are both of the AA genotype and both of the AG genotype for mating; or select the male parent and the female parent that are both of the AG genotype for mating, and continue to screen out individuals with the AA genotype in the offspring of the mating.

[0110] As much as possible, screen out Litopenaeus vannamei individuals with the growth-advantageous AA / AA homozygous type, and exclude Litopenaeus vannamei individuals with slow growth as early as possible, so as to increase the aquaculture yield of Litopenaeus vannamei.

[0111] Example 2

[0112] Perform further functional verification on the PDE11A gene (SEQ ID NO.1) where the SNP molecular marker combination of Litopenaeus vannamei in Example 1 is located, specifically including the following steps:

[0113] S1. Cultivation of Litopenaeus vannamei

[0114] Cultivate Litopenaeus vannamei in a recirculating seawater system at a temperature of 27°C ± 1°C. Feed commercial feed pellets (available on the market) at 5% of the body weight per day, and feed in three portions. At the same time, change the culture water once a day. Select 120 Litopenaeus vannamei with a body weight of 5.33 g ± 1.16 g and a body length of 7.43 g ± 0.57 cm as test materials. At this time, Litopenaeus vannamei is in the larval stage.

[0115] S2. RNA interference of Litopenaeus vannamei

[0116] (1)Divide the above 120 cultured Litopenaeus vannamei into 3 equal parts on average, set up a blank control group (PBS), a negative control group (siNC), and an experimental group (siPDE11A), with 40 tails in each group.

[0117] (2)Use the siDirect v2.1 tool to design siRNA sequences targeting the PDE11A gene. Among them,

[0118] Primer design for the negative control group:

[0119] siNC-F: 5’-UUCUCCGAACGUGUCACGU-3’ (SEQ ID NO.12)

[0120] siNC-R: 5’-ACGUGACACGUUCGGAGAA-3’ (SEQ ID NO.13)

[0121] Primer design for the experimental group:

[0122] siPDE11A-F: 5'-CCGCAUCAUGACACAUACA-3' (SEQ ID NO.14)

[0123] siPDE11A-R: 5'-UGUAUGUGUCAUGAUGCGG-3' (SEQ ID NO.15)

[0124] The above 4 primers (SEQ ID NO.10 - 13) were delivered to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for primer sequence synthesis.

[0125] (3) After centrifugation, the primers synthesized from the above SEQ ID NO.12 - 15 sequences were dissolved with 1.5×PBS, and a blank control group and an experimental group were set up respectively.

[0126] Blank control group (PBS): Each Litopenaeus vannamei was injected with 20 μL of 1.5×PBS; Negative control group (siNC): Each Litopenaeus vannamei was injected with siNC-F and siNC-R dissolved in 1×PBS, and the injection dose was 2 μg / g / shrimp for both siNC-F and siNC-R (that is, each Litopenaeus vannamei was injected with 2 μg according to each g of body weight, and so on, and the injection dose was increased according to the body weight of each shrimp);

[0127] Experimental group (siPDE11A): Each Litopenaeus vannamei was injected with siPDE11A-F and siPDE11A-R dissolved in 1.5×PBS, and the injection dose was 2 μg / g / shrimp for both siPDE11A-F and siPDE11A-R (same as the negative control group); Injection was performed once every 4 days, targeting the middle part of the second to third abdominal segments. The experiment lasted for 22 days. After the experiment, the hepatopancreas and muscle samples of all Litopenaeus vannamei were collected and stored in a -80°C refrigerator, and the body length of each Litopenaeus vannamei was measured (accurate to mm) before each injection and before sample collection.

[0128] The measurement results are as Figure 8 shown. The body length increase of Litopenaeus vannamei in the experimental group was significantly lower than that in the blank control group (PBS) and the negative control group (siNC). The body length increase of Litopenaeus vannamei in the experimental group was about 1 cm, while the body length increase in the blank control group (PBS) and the negative control group (siNC) was mainly above 1 cm, and the body length increase of Litopenaeus vannamei in the blank control group (PBS) was the largest, reaching 1.5 cm.

[0129] S3. Determination of the relative expression level of the PDE11A gene in Litopenaeus vannamei

[0130] (1) Extraction of total RNA from Litopenaeus vannamei

[0131] Total RNA was extracted from the hepatopancreas and muscle of Litopenaeus vannamei collected above by the Trizol method, and the concentration and quality of the extracted RNA were evaluated by Nanodrop 2000 and 1.5% agarose gel electrophoresis. The specific experimental steps for extracting RNA by the Trizol method are as follows:

[0132] 1) After washing experimental instruments such as pipette tips, eight-well strips, grinding magnetic beads, scissors, and forceps, sterilize them in an autoclave and then put them in an oven for use;

[0133] 2) Cut 50 micrograms of Litopenaeus vannamei tissue (hepatopancreas or muscle tissue) and put it into a 1.5 ml RNase-free centrifuge tube, and soak the tissue with 500 μL of Trizol;

[0134] 3) Add 3 - 5 grinding magnetic beads to the centrifuge tube, place the centrifuge tube in a tissue grinder for grinding, and the grinding conditions are 70 Hz and 4°C for 3 min;

[0135] 4) After grinding the tissue, add 500 μL of Trizol to the centrifuge tube, mix well and place on ice for 5 min;

[0136] 5) Centrifuge at 12000 rpm and 4°C for 5 min;

[0137] 6) Take the supernatant to a new centrifuge tube, add about 200 μL of chloroform, shake vigorously for about 15 s to mix well, and place on ice for 15 min;

[0138] 7) Centrifuge at 12000 rpm and 4°C for 15 min;

[0139] 8) Aspirate the supernatant into another centrifuge tube, add an equal volume of isopropanol pre-cooled at -20°C, mix gently, and place in an environment at -20°C for 2 h;

[0140] 9) Centrifuge at 1200 rpm and 4°C for 10 min;

[0141] 10) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, shake gently to suspend the precipitate, and centrifuge at 10000 rpm and 4°C for 5 min;

[0142] 11) Discard the supernatant, add 1 mL of pre-cooled absolute ethanol, shake gently to suspend the precipitate, and centrifuge at 10000 rpm and 4°C for 5 min;

[0143] 12) Discard the supernatant, open the centrifuge tube cap and air-dry naturally in a laminar flow cabinet for 5 - 10 min;

[0144] 13) Add about 50 μL of DEPC water to dissolve the precipitate;

[0145] 14) Take 1 μL of the RNA sample and measure its concentration and purity using a Nanodrop 2000 micro-spectrophotometer;

[0146] 15) Take 1 μg of the RNA sample and detect its integrity using 1.5% agarose gel electrophoresis, and observe whether there are obvious 28S and 18S rRNA bands;

[0147] 16) Store the qualified RNA sample in a -80 °C refrigerator.

[0148] Among them, the results of the total RNA extraction from the hepatopancreas after RNA interference are as Figure 9 shown in a, where M is the Marker, 1 - 12 are the total RNAs from the hepatopancreas after RNA interference, and there is a relatively bright band at about 500 bp of 28S;

[0149] The results of the total RNA extraction from muscle are as Figure 9 shown in b, where M is the Marker, 1 - 12 are the total RNAs from muscle after RNA interference, and there is a relatively bright band at about 500 bp of 28S. It shows that the quality of the total RNA extraction is good and can be used for subsequent experiments.

[0150] (2) Reverse transcription

[0151] Using the total RNA of Litopenaeus vannamei extracted above as a template, reverse transcription was carried out using an All-In-One 5X RT MasterMix kit (the following reagents are all included in the kit). The reverse transcription system was: 4 μL of 5X All-In-One RT MasterMix, 1 μg of total RNA, and made up to 20 μL with RNase-free water; the reverse transcription program was: 15 min at 37 °C, 10 min at 60 °C, 3 min at 95 °C, and stored at 4 °C to obtain the cDNA of Litopenaeus vannamei.

[0152] (3) qRT-PCR

[0153] Using the cDNA of Litopenaeus vannamei obtained above diluted 5 times as a template, with 18S rRNA as the internal reference gene, specific primers for the PDE11A gene and the internal reference gene were designed through NCBI. Among them,

[0154] Forward primer 18S-F: 5’-TATACGCTAGTGGAGCTGGAA-3’ (SEQ ID NO.16);

[0155] Reverse primer 18S-R: 5’-GGGGAGGTAGTGACGAAAAAT-3’ (SEQ ID NO.17);

[0156] Forward primer PDE11A-F: 5’-ATGCTGGACCTTGCACGAAT-3’ (SEQ ID NO.18);

[0157] Reverse primer PDE11A-R: 5’-TTGATGGGGAAGCGTGACTC-3’ (SEQ ID NO.19).

[0158] The BlasTaq 2X qPCR MasterMix kit was used for qRT-PCR to detect the expression level of the target gene. The reaction system for qRT-PCR was as follows:

[0159] 2 μL of cDNA from Litopenaeus vannamei,

[0160] 10 μL of BlasTaq 2X qPCR MasterMix,

[0161] 0.5 μL of forward primer (10 μM);

[0162] 0.5 μL of reverse primer (10 μM),

[0163] 7 μL of DEPC water.

[0164] Three biological replicates were set for each sample, and four technical replicates were set for each biological replicate. The samples were loaded in a 96-well plate, centrifuged at 3000 rpm for 1 min, and qRT-PCR was performed using a fluorescence quantitative gene amplifier qTOWER3G. The reaction program for qRT-PCR was as follows:

[0165] Pre-denaturation at 95℃ for 30 s,

[0166] Denaturation at 95℃ for 10 s,

[0167] Annealing and extension at 60℃ for 30 s, with a total of 40 cycles from denaturation to annealing and extension.

[0168] The specificity of the primers was verified by melting curve, and the relative expression level of the target gene was calculated using the 2- △△t method.

[0169] The results of qRT-PCR were as Figure 10As shown, whether in the hepatopancreas or muscle tissue of Litopenaeus vannamei, the relative expression levels of the PDE11A gene in the experimental group were lower than those in the control group (blank control group and negative control group). Among them, in the hepatopancreas of Litopenaeus vannamei, the relative expression levels of the PDE11A gene in the experimental group were significantly lower than those in the control group; in the muscle of Litopenaeus vannamei, the relative expression levels of the PDE11A gene in the experimental group were extremely significantly lower than those in the control group. The results showed that siPDE11A effectively inhibited the expression of the PDE11A gene and had a significant impact on the growth traits of Litopenaeus vannamei.

[0170] In summary, the PDE11A gene of Litopenaeus vannamei can regulate the growth rate of Litopenaeus vannamei. By transiently treating Litopenaeus vannamei at the larval stage through RNA interference technology, it was found that siPDE11A significantly and extremely significantly inhibited the expression of the PDE11A gene in the hepatopancreas and muscle tissues respectively, reduced the expression waste of the PDE11A gene of Litopenaeus vannamei at the larval stage, and could narrow the growth rate differences among individuals of Litopenaeus vannamei in the population in a short time; after restoring the expression of the PDE11A gene of Litopenaeus vannamei, overexpressing the PDE11A gene can be used to accelerate the growth of individual Litopenaeus vannamei, improve the breeding efficiency of Litopenaeus vannamei, and increase the economic value.

[0171] Comparative Example 1

[0172] The difference between this comparative example and Example 1 is that the primers used are:

[0173] Trmt10a-F: 5’-CGGTGTTCATAAAGAGGATAC-3’ (SEQ ID NO.20);

[0174] Trmt10a-R: 5’-CTCTCCACTTTTGTGTCGTTC-3’ (SEQ ID NO.21).

[0175] Among them, the primers used in this comparative example are the publicly reported primers for detecting SNP loci for the rapid growth rate of Litopenaeus vannamei.

[0176] It was found that the primers provided in this Comparative Example 1 could not detect the SNP molecular marker combination in Example 1, and using the primer set provided by the present invention for PCR reaction could quickly detect the SNP molecular marker combination in Example 1.

[0177] Therefore, it can be concluded that through genome-wide association study, two SNP molecular marker loci related to body length located on the PDE11A gene of Litopenaeus vannamei are identified, namely the SNP molecular marker combination, which are respectively named PDE11A-2877 (the first SNP locus) and PDE11A-8932 (the second SNP locus). Among them, the sequence of the PDE11A-2877 SNP molecular marker is shown in SEQ ID NO.2, the SNP locus is located at the 14th position from the 5'-end of the sequence shown in SEQ ID NO.2, and the polymorphism is of the A / G type; the sequence of the PDE11A-8932 SNP molecular marker is shown in SEQ ID NO.3, the SNP locus is located at the 217th position from the 5'-end of the sequence shown in SEQ ID NO.3, and the polymorphism is of the A / G type; the primers SEQ ID NO.4 and SEQ ID NO.5 designed according to the SEQ ID NO.2 sequence can detect the PDE11A-2877 SNP molecular marker, and the primers SEQ ID NO.6 and SEQ ID NO.7 designed according to the SEQ ID NO.3 sequence can detect the PDE11A-8932 SNP molecular marker. The above primers can also be prepared into a kit. Therefore, the two SNP molecular markers PDE11A-2877 and PDE11A-8932 provided by the present invention can be used to assist in the selection of fast-growing parents. By selecting parents with the AA / AA genotype in this SNP molecular marker combination, the growth rate of the breeding population can be increased, providing an innovative tool for the molecular breeding of Litopenaeus vannamei, shortening the breeding cycle, and facilitating industrial development and efficiency improvement.

[0178] The terms and expressions used herein are for descriptive purposes only, and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean excluding any equivalent features of the illustration and description (or parts thereof). It should be recognized that various modifications may also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be regarded as covering all such equivalents.

[0179] Similarly, it should be noted that although the present invention has been described with reference to the current specific embodiments, those of ordinary skill in the art in this technical field should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications of the above embodiments are within the scope of the spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. Application of a reagent for detecting a combination of SNP molecular markers in the breeding of long body length or fast growth rate individuals of Litopenaeus vannamei, characterized in that: The SNP molecular marker combination includes a first SNP molecular marker and a second SNP molecular marker; The first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2; the polymorphism of the first SNP molecular marker is A / G; The second SNP molecular marker is located at the 217th position from the 5' end of the sequence shown in SEQ ID NO.3; the polymorphism of the second SNP molecular marker is A / G.

2. The use of the reagent for detecting the combination of SNP molecular markers according to claim 1 in the breeding of long body length or fast growth rate of Penaeus vannamei, characterized in that: Specifically, the reagent for detecting the SNP molecular marker combination is used to screen out the AA / AA homozygous Penaeus vannamei individuals with growth advantage, and exclude the Penaeus vannamei individuals with slow growth.

3. Use of the reagent for detecting the combination of SNP molecular markers according to claim 1 in the breeding of long body length or fast growth rate of Penaeus vannamei, characterized in that: The reagent is a primer set, and the primer set comprises the following base sequence: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3'; PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3'; PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3'; PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3'; Wherein, the first SNP molecular marker is detected by using the PDE11A-2877-Fw and the PDE11A-2877-Rw; and the second SNP molecular marker is detected by using the PDE11A-8932-Fw and the PDE11A-8932-Rw.

4. Use of the reagent for detecting the combination of SNP molecular markers according to claim 1 in the breeding of long body length or fast growth rate of Penaeus vannamei, characterized in that: The reagent is a detection kit comprising a primer set, and the primer set comprises the following base sequence: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3'; PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3'; PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3'; PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3'; Wherein, the first SNP molecular marker is detected by using the PDE11A-2877-Fw and the PDE11A-2877-Rw; and the second SNP molecular marker is detected by using the PDE11A-8932-Fw and the PDE11A-8932-Rw.

5. A method for breeding Penaeus vannamei using a combination of SNP molecular markers, characterized in that: The following steps are involved: Step A: detecting the SNP molecular marker combination of Litopenaeus vannamei to obtain the genotype of the SNP combination site; the SNP molecular marker combination includes a first SNP molecular marker and a second SNP molecular marker; The first SNP molecular marker is located at the 14th position from the 5' end of the sequence shown in SEQ ID NO.2; the polymorphism of the first SNP molecular marker is A / G; The second SNP molecular marker is located at position 217 from the 5' end of the sequence shown in SEQ ID NO.3; the polymorphism of the second SNP molecular marker is A / G; Step B: screening is performed according to the genotype of the SNP combination site; the genotype of the SNP combination site is AA / AA, AA / AG, AA / GG, AG / AG, AG / GG or GG / GG; the screening basis is the genotype-body length rule; Step C: breeding Penaeus vannamei with the genotype of the SNP combination site being AA / AA genotype.

6. The method for breeding Penaeus vannamei according to claim 5, characterized in that: The step A comprises: Step A-1: ​​extracting genomic DNA of the shrimp Penaeus vannamei; Step A-2: Using the genomic DNA of the vannamei shrimp as a template, PCR amplification was performed using the following primer pairs: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3'; PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3'; PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3'; PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3'; The ratio of the PCR amplification system is: 1-2 μL of genomic DNA of Penaeus vannamei with a concentration of 100 ng / μL; 1-2 μL of forward primer Fw; 1-2 μL of reverse primer Rw, wherein the concentrations of the forward primer Fw and the reverse primer Rw are both 10 μM; 10-13 μL of 2×Taq Plus Master Mix Ⅱ Plus; 8-10 μL of enzyme-free sterile water; The PCR amplification procedure includes: pre-denaturation at 95°C for 3 min; then 32 cycles of: 95°C for 15 s; 56-58°C for 20-30 s; 72°C for 40 s; and full extension at 72°C for 10 min to obtain a PCR amplification product; Step A-3: Sequencing the PCR amplification product to obtain the genotype of the SNP combination site.

7. The method for breeding Penaeus vannamei according to claim 5, characterized in that: The screening basis is the body length of the vannamei shrimp: AA / AA genotype>AA / AG genotype>AA / GG genotype>AG / AG genotype>AG / GG genotype>GG / GG genotype.

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