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

Abstract

The invention discloses a PDE11A gene of litopenaeus vannamei, an SNP (Single Nucleotide Polymorphism) molecular marker combination, a detection primer and application of the PDE11A gene. The nucleotide sequence of the PDE11A gene of the litopenaeus vannamei is SEQ ID NO. 1. The SNP molecular marker combination is located on the SEQ ID NO.1 sequence, and the SNP molecular marker combination comprises a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at the fourteenth site from the 5'end of a sequence shown as SEQ ID NO.2, and the polymorphism is A / G; the second SNP molecular marker is located at the 217th site from the 5'end of the sequence shown as SEQ ID NO.3, and the polymorphism is A / G. The AA / AA genotype in the SNP molecular marker combination is used for selecting parents, so that the growth speed of a breeding population can be increased, an innovative tool is provided for molecular breeding of litopenaeus vannamei, the breeding period is shortened, and industrial development is assisted.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and specifically relates to a combination of a PDE11A gene and a SNP molecular marker of a shrimp Penaeus vannamei, a detection primer and an application thereof. Background Art

[0002] Penaeus vannamei, also known as the South American white shrimp and Pacific white shrimp, belongs to the Arthropoda, Crustaeea, Decapoda, Penaeidae, and Penaeus genus. It is a pillar species of the global aquaculture industry, with the highest aquaculture yield and one of the highest single aquaculture species. Penaeus vannamei is widely cultivated due to its fast growth rate, strong environmental adaptability and high economic value. However, during the cultivation process, there are significant differences in the growth performance of Penaeus vannamei. The growth traits of shrimp are mainly affected by a variety of genetic and environmental factors. Among them, body weight and growth rate are important indicators to measure its economic value. Light weight or slow growth rate will seriously affect the breeding efficiency and economic benefits.

[0003] Molecular breeding of Penaeus 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 individuals with better performance for reproduction, but because growth traits have strong quantitative and environmental influences, the breeding cycle is long and the efficiency is low, which makes it difficult to meet the growing market demand; (2) Lack of accurate molecular markers: Although some genomic technologies (such as genomic selection and molecular marker-assisted selection) have been applied to shrimp breeding, there is still a lack of efficient molecular markers for growth traits. Existing markers are often generalized and cannot accurately identify genetic factors that are highly correlated with growth traits, resulting in limited efficiency of genetic improvement; (3) Difficulty in achieving accuracy of genetic improvement: Due to the complex genetic basis of growth traits of Penaeus vannamei, traditional phenotypic selection methods are often greatly affected by environmental factors, making it difficult to achieve accurate 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 on the genome. As the third-generation molecular marker, SNP molecular marker is the most widely used and latest molecular marker. It has the characteristics of high density, strong stability, and co-dominance. It is currently the most widely used molecular marker technology in economic crustaceans such as shrimp and crab. However, as a complex quantitative trait, the growth traits of Penaeus vannamei still need to be further explored and verified with new genetic markers.

[0005] Phosphodiesterase 11A gene (PDE11A) is a member of the phosphodiesterase (PDE) superfamily. This gene encodes a bifunctional 3',5'-cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterase that can catalyze the degradation of cAMP and cGMP into the corresponding 5'-monophosphates, thus playing an important role in cell signaling. By regulating the cAMP and cGMP signaling pathways, PDE11A has potential functional regulatory effects in a variety of biological processes. In the shrimp Litopenaeus vannamei, the PDE11A gene has not been reported, and no studies have explored the relationship between this gene and shrimp growth traits.

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

[0007] The purpose of the present invention is to provide a growth trait PDE11A gene, a SNP molecular marker combination, and a detection primer for Penaeus vannamei, and to apply them to the breeding of fast-growing shrimp varieties, accelerate the genetic improvement of growth traits, and improve breeding efficiency and economic benefits. In order to achieve the above purpose: In one aspect, the present invention provides a PDE11A gene of Penaeus vannamei. The nucleotide sequence of the PDE11A gene of Penaeus vannamei is shown in SEQ ID NO.1.

[0008] Another aspect of the present invention provides an application of the PDE11A gene of Penaeus vannamei in breeding of Penaeus vannamei.

[0009] On the other hand, the present invention provides a SNP molecular marker combination, the sequence of the SNP molecular marker combination is 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.

[0010] Another aspect of the present invention provides an application of a SNP molecular marker combination in breeding of Penaeus vannamei.

[0011] Furthermore, the breeding of Penaeus vannamei includes body length screening, individual identification of broodstock, group selection or strain maintenance of Penaeus vannamei.

[0012] Another aspect of the present invention provides a primer set, which comprises one, a pair or more of the following base sequences: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3'; PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3'; PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3'; PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3'; Further, the SNP molecular marker combination is detected using a primer set, and the sequence of the SNP molecular marker combination is 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 the first SNP molecular marker is detected using PDE11A-2877-Fw and PDE11A-2877-Rw; 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 the second SNP molecular marker is detected using PDE11A-8932-Fw and PDE11A-8932-Rw.

[0013] Another aspect of the present invention provides an application of a primer set in breeding of Penaeus vannamei.

[0014] Another aspect of the present invention provides a detection kit comprising a primer set.

[0015] Another aspect of the present invention provides an application of a detection kit in breeding of Penaeus vannamei.

[0016] On the other hand, the present invention provides a method for breeding Penaeus vannamei using a SNP molecular marker combination, comprising the following steps: step A: detecting the SNP molecular marker combination of Penaeus vannamei to obtain the genotype of the SNP combination site; step B: screening 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 genotype; the screening basis is the genotype-body length rule; step C: breeding Penaeus vannamei with the genotype of the SNP combination site being the AA / AA genotype.

[0017] Further, step A comprises: step A-1: ​​extracting genomic DNA of Penaeus vannamei; step A-2: using the genomic DNA of Penaeus vannamei as a template, performing PCR amplification 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 (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 sterile water; the PCR amplification procedure includes: pre-denaturation at 95℃ / 3 min; then 32 cycles: 95℃ / 15 s; 56-58℃ / 20-30 s; 72℃ / 40 s; fully extended at 72℃ / 10min to obtain PCR amplification products; step A-3: sequencing the PCR amplification products to obtain the genotype of the SNP combination site; the genotype of the SNP combination site is SEQ A / G at position 2877 and A / G at position 8932 of the sequence shown in IDNO.1; A / G represents that there is A / G polymorphism at this SNP site, and the A genotype is superior to the G genotype.

[0018] Furthermore, the screening basis is the body length of Penaeus vannamei: the body length of Penaeus vannamei with SNP combination site genotype of AA / AA genotype>the body length of Penaeus vannamei with SNP combination site genotype of AA / AG genotype>the body length of Penaeus vannamei with SNP combination site genotype of AA / GG genotype>the body length of Penaeus vannamei with SNP combination site genotype of AG / AG genotype>the body length of Penaeus vannamei with SNP combination site genotype of AG / GG genotype>the body length of Penaeus vannamei with SNP combination site genotype of GG / GG genotype.

[0019] The present invention locates two SNP molecular marker sites associated with body length on the PDE11A gene of Penaeus vannamei by whole genome association analysis, namely a SNP molecular marker combination, which are 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. The AA / AA genotype in this SNP molecular marker combination is used to select parents, which can increase the growth rate of the breeding population, provide innovative tools for the molecular breeding of Penaeus vannamei, shorten the breeding cycle, and help industry development and improve efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed for protection.

[0021] Figure 1 The Manhattan plot of the genome-wide association analysis of the body length of Penaeus vannamei in Example 1; Figure 2is an agarose gel electrophoresis diagram of the genomic DNA of Penaeus vannamei in Example 1 (wherein, M is a marker; A is the genomic DNA of Penaeus vannamei numbered 1-12; B is the genomic DNA of Penaeus vannamei numbered 13-24, all of which are more than 5000 bp in length and have good integrity); Figure 3 The agarose gel electrophoresis diagram of the PCR amplification products of SEQ ID NO. 2 and SEQ ID NO. 3 of Litopenaeus vannamei in Example 1 (wherein M is a marker; 1-12 in a are PCR products of the PDE11A-2877 site, with a brighter band at around 441 bp; 1-12 in b are PCR products of the PDE11A-8932 site, with a brighter band at around 692 bp); Figure 4 This is a diagram illustrating the sequencing process in Example 1; Figure 5 This is the reverse sequencing peak diagram of the PCR amplification product of SEQ ID NO.2 in Example 1 (SEQ ID NO.10 has a polymorphic base at position 289); Figure 6 This is the forward sequencing peak diagram of the PCR amplification product of SEQ ID NO.3 in Example 1 (SEQ ID NO.11 has a polymorphic base at position 154); Figure 7 The body length distribution of Penaeus vannamei individuals with different genotypes in Example 1 (wherein the abscissa represents the genotype, which is AA / AA, AA / AG, AA / GG, AG / AG, AG / GG and GG / GG in sequence; the ordinate represents the body length of Penaeus vannamei, in (mm); "***" represents that the P value has a significant difference in the body length of Penaeus vannamei individuals between the two genotypes); Figure 8 This is a graph showing the growth of the body length of Penaeus vannamei after RNA interference in Example 2 (wherein the abscissa is the growth of body length, in cm; the ordinate is different RNA interference conditions, green is the blank control group (PBS), purple is the negative control group (siNC), and orange is the experimental group (siPDE11A)); Fig. 9 Agarose gel electrophoresis of total RNA from hepatopancreas and muscle of Litopenaeus vannamei after RNA interference (M stands for Maker, 1-12 in a are total RNA from hepatopancreas after RNA interference, with a brighter band at around 500 bp; 1-12 in b are total RNA from muscle after RNA interference, with a brighter band at around 500 bp); Fig.10This is the relative expression level of the PDE11A gene in the hepatopancreas and muscle of Penaeus vannamei after RNA interference (the horizontal axis is the hepatopancreas and muscle; the vertical axis is the relative expression level; green is the blank control group (PBS), purple is the negative control group (siNC), and orange is the experimental group (siPDE11A); "***" represents that the P value has a significant difference in the individual body length of Penaeus vannamei between the two genotypes). DETAILED DESCRIPTION

[0022] The detailed features and advantages of the present invention are described in detail in the specific implementation modes below, and the contents are sufficient to enable any person skilled in the art to understand the technical contents of the present invention and implement them accordingly. Moreover, according to the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant objects and advantages of the present invention.

[0023] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0025] (1) Source of sample materials In this embodiment, the shrimp Penaeus vannamei was purchased from Hainan Renhai Aquatic Technology Co., Ltd. (Wenchang City, Hainan Province).

[0026] (2) Sources of reagents and consumables Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0027] (3) Source of instruments and equipment Table 2 Sources and models of instruments and equipment required for the experiment

[0028] Example 1

[0029] A SNP molecular marker combination associated with the body length growth trait of Penaeus vannamei comprises the following steps: S1. Sample collection and sequencing. The specific steps are as follows: (1) 996 individuals were randomly selected from a population of 8-month-old Penaeus vannamei. The body length of each individual was measured with a ruler (accurate to 1 mm), and the antennae were collected for genome analysis. The antennae samples of Penaeus vannamei were sent to Beijing Novogene Technology Co., Ltd. for genome resequencing, and a total of 14,276,173 SNP loci were obtained. After quality control of the 14,276,173 SNP loci obtained above, a total of 2,706,353 SNP loci were screened.

[0030] (2) Based on the gene resequencing results in step (1) above, the whole genome SNP genotyping data of the 2,706,353 SNP sites that have been screened were used to perform principal component analysis (PCA) and population structure analysis using PLINK software. The principal components, population structure matrix, and sex were used as covariates to control the interference of population structure and sex on the analysis results. The compressed mixed linear model (CMLM) of the GAPIT package was used to combine the SNP genotype and phenotype data of Penaeus vannamei for a genome-wide association study (GWAS).

[0031] The formula is as follows: , Where Y is an n×1 phenotype vector containing the phenotypic values ​​of n individuals. X is an n×p design matrix, which includes independent variables of fixed effects, such as SNP genotypes and other covariates (gender, age, etc.). β is a p×1 fixed effect coefficient vector, which represents the fixed effect of SNP genotype on phenotype. Zc is an n×q compressed design matrix used to represent random effects. uc is a q×1 random effect vector, which is usually assumed to follow a multivariate normal distribution uc~N (0, Iq), where Iq is the q×q identity matrix, is the variance of the random effect. is an n×1 error vector, assuming ~N(0, In), where In is the n×n identity matrix, is the variance of the error.

[0032] According to Bonferroni correction α Bonferroni =α / m, where α is the global significance level (usually 0.05) and m is the number of independent tests performed (usually the number of SNPs tested).

[0033] The results are as follows Figure 1 As shown in Figure 2, the significance threshold of the association between SNP and body length trait of Penaeus vannamei determined by GWAS analysis was 1.85×10 -8, the SNP sites associated with GWAS analysis were sorted according to their P values, and unique SNP sites significantly associated with the growth traits of Penaeus vannamei body length were screened out. The results showed that there were two SNP molecular marker sites on the PDE11A gene (LOC113813671; SEQID NO.1) of Penaeus vannamei, namely the SNP molecular marker combination, named PDE11A-2877 (the first SNP site) and PDE11A-8932 (the second SNP site). Among them, PDE11A-2877 is located at position 2887 on chromosome 37 of Penaeus vannamei, and the nucleotide sequence of PDE11A-2877 is shown in SEQ ID NO.2. Through genotyping, it was found that the genotype of PDE11A-2877 was 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 position 8932 on chromosome 37 of Litopenaeus vannamei. The nucleotide sequence of PDE11A-8932 is shown in SEQ ID NO.3. Through genotyping, it was found that the genotype of PDE11A-8932 is 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.

[0034] S2. Extraction of genomic DNA from Litopenaeus vannamei To further verify the genotype with obvious advantages, 200 large and small shrimps were randomly selected from the 9-month-old Penaeus vannamei population, and the antennae of the Penaeus vannamei were cut and ground into powder using liquid nitrogen. The Marine Animal Genome Extraction Kit (Tiangen Biochemical, the following reagents and consumables are all from this kit) was used to extract the genomic DNA of the Penaeus vannamei. The DNA extraction steps are as follows: (1) Sample pretreatment: Weigh 30 mg of shrimp antennae, add 200 μL of GA buffer into a centrifuge tube, mix gently, and shake for 15 seconds to ensure that the shrimp antennae tissue is completely infiltrated.

[0035] (2) Proteinase K digestion: Add 20 mL of Proteinase K solution (20 mg / mL) to the centrifuge tube, mix thoroughly, and incubate in a 56°C incubator until the tissue is completely dissolved.

[0036] (3) Lysis and precipitation: Add 200 μL of GB lysis buffer, mix thoroughly, and place in a 70°C water bath for 10 minutes. Then add 200 μL of anhydrous ethanol and mix well.

[0037] (4) Purification by adsorption column: The mixed solution was transferred to the CB3 adsorption column, placed in a collection tube, centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. Then, 500 μL of GD buffer and 600 μL of PW rinse solution were added, and each centrifuged for 30 seconds to clean the adsorption column to remove impurities.

[0038] (5) Drying the adsorption column: Place the cleaned adsorption column in the 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.

[0039] (6) DNA elution: Place the adsorption column in a new centrifuge tube, add 50-200 μL of sterile water to the middle of the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the eluted DNA solution.

[0040] (7) Quality testing and preservation: Use a spectrophotometer to test the concentration and purity of DNA to ensure OD 260 / OD 280 Between 1.8-2.0, OD 260 / OD 230 Greater than 2.0.

[0041] The final concentration of the obtained Penaeus vannamei genomic DNA was 100 ng / μL. The Penaeus vannamei genomic DNA was detected by 1.5% agarose gel electrophoresis (24 Penaeus vannamei genomic DNAs were randomly selected for detection), and the remaining Penaeus vannamei genomic DNA was stored in a -20℃ refrigerator for later use.

[0042] The results of agarose gel electrophoresis of the genomic DNA of Penaeus vannamei are as follows Figure 2 As shown, M is a marker, 1-24 are target genes (Penaeus vannamei genomic DNA), all of which are longer than 5000 bp and have good integrity.

[0043] S3. Primer design The NCBI primer design function module Primer-BLAST was used to design primers for amplification of the PDE11A-2877 site and PDE11A-8932 site of Penaeus vannamei: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3' (SEQ ID NO.4) PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3' (SEQ ID NO.5) PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3' (SEQ ID NO.6) PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3' (SEQ ID NO.7) The four synthesized primer sequences (SEQ ID NO.4-7) were sent to Beijing Qingke Biotechnology Co., Ltd. for primer synthesis and used for the amplification of SEQ ID NO.2 and SEQ ID NO.3. Among them, SEQ ID NO.4-5 were used for the amplification of SEQ ID NO.2, and SEQ ID NO.6-7 were used for the amplification of SEQ ID NO.3.

[0044] S4. PCR amplification reaction Using the genomic DNA of Penaeus vannamei obtained in step S2 as a template, the PDE11A-2877 and PDE11A-8932 primers synthesized in step S3 are used to perform PCR amplification reactions respectively. At the same time, the synthesized PDE11A-2877 and PDE11A-8932 primers are placed in a kit to make a detection kit, which can also perform PCR amplification reactions. The specific steps are as follows: (1) The PCR amplification reaction system (25 μL system) is designed as follows: Litopenaeus vannamei genomic DNA (100 ng / μL), 1 μL; Forward primer Fw (10 μM), 1 μL; Reverse primer Rw (10 μM), 1 μL; 2×Taq Plus Master Mix Ⅱ (Plus), 12.5 μL; Enzyme-free sterile water, 9.5 μL.

[0045] (2) The PCR amplification reaction procedure is: Pre-denaturation at 95°C for 3 min; Denaturation at 95°C for 15 s; Annealing at 56 °C for 20 s; Extension at 72°C for 40 s, with a total of 32 cycles from denaturation to extension; Full extension at 72°C for 10 min; Store at 4°C.

[0046] After the PCR amplification reaction was completed, the PCR amplification products of the PDE11A-2877 site and the PCR products of the PDE11A-8932 site were obtained respectively, and the PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0047] The agarose gel electrophoresis results of the PCR amplification product of SEQ ID NO.2 are as follows Figure 3 As shown in a, M is a marker, 1-12 are PCR products of the PDE11A-2877 site, and there is a brighter band at around 441 bp.

[0048] The agarose gel electrophoresis results of the PCR amplification product of SEQ ID NO.3 are as follows Figure 3 As shown in b, M is a marker, 1-12 are the PCR products of the PDE11A-8932 site, and there is a brighter band at around 692 bp.

[0049] S5. Verification of SNP sites (1) The PCR amplification product obtained in step S4 was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis. The sequencing process is shown in the figure below. Figure 4 As shown, the PCR amplification product of SEQ ID NO.2 was reverse sequenced, and the reverse sequencing primer T1 was: 5'-GTTTCAGAACACACGAGTAAAG-3' (SEQ ID NO.8); the PCR amplification product of SEQ ID NO.3 was forward sequenced, and the forward sequencing primer T2 was: 5'-ATTCCAGAAAGTGAAGATACCC-3' (SEQ ID NO.9), and the sequencing peak diagram of the PCR amplification product was obtained ( Figure 5 , Figure 6 ).

[0050] (2) According to the sequencing peak diagram of the PCR amplification product obtained above ( Figure 5 , Figure 6 ) were used to genotype the two SNP loci.

[0051] The reverse sequencing result of SEQ ID NO.2 is shown as 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 shorter 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.

[0052] The forward sequencing result of SEQ ID NO.3 is shown as SEQ ID NO.11 (ZX1). The sequence of SEQ ID NO.11 is a partial sequence of SEQ ID NO.3. In SEQ ID NO.11, if there is only one A base peak at the 154th position from the 5' end, 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.

[0053] (3) One-way analysis of variance (ANOVA) was used to detect the differences in body length of the three genotypes AA, AG and GG of the SNP loci of Penaeus vannamei. The results are shown in Tables 3 and Figure 7 As shown in the figure, through the statistical analysis of the individual genotypes of Penaeus vannamei, it was found that the individuals with the genotype of PDE11A-2877 and PDE11A-8932 of AA / AA grew significantly faster than the individuals with other genotypes under the same culture conditions; the individuals with the genotype of PDE11A-2877 and PDE11A-8932 of GG / GG grew significantly slower than the individuals with other genotypes under the same culture conditions. These results highlight that the genotype of PDE11A-2877 and PDE11A-8932 of AA / AA has a clear advantage in promoting the rapid growth of Penaeus vannamei, and may be an important genetic marker affecting growth traits.

[0054] Table 3 Correlation between SNP markers of PDE11A gene and body length of Penaeus vannamei

[0055] The SNP typing results can be used for individual identification of broodstock, group breeding and strain maintenance: (1) Keep the male or female parent whose SNP molecular marker combination (the first SNP molecular marker and the second SNP molecular marker) is AA type for seed preservation and generation; (2) If the father and mother are not both of the AA genotype, then select the two SNP sites of the father and mother that are both of the AA genotype and the AG genotype for mating; or select the father and mother that are both of the AG genotype for mating, and continue to screen out individuals with the AA genotype from the mating offspring.

[0056] Try to screen out AA / AA homozygous Penaeus vannamei individuals with growth advantages as much as possible, and eliminate slow-growing Penaeus vannamei individuals as early as possible, thereby increasing the farming yield of Penaeus vannamei.

[0057] Example 2

[0058] The PDE11A gene (SEQ ID NO. 1) where the SNP molecular marker combination of Litopenaeus vannamei in Example 1 is located is further functionally verified, specifically comprising the following steps: S1. Farming of Penaeus vannamei The shrimp were cultured in a circulating seawater system at 27°C ± 1°C, and were fed with commercial feed pellets (available on the market) at 5% of their body weight three times a day, and the culture water was changed once a day. 120 shrimps weighing 5.33g ± 1.16 g and 7.43g ± 0.57 cm in length were selected as experimental materials. At this time, the shrimps were in the larval stage.

[0059] S2. RNA interference in Litopenaeus vannamei (1) The 120 cultured Penaeus vannamei were equally divided into three groups: a blank control group (PBS), a negative control group (siNC), and an experimental group (siPDE11A), with 40 shrimp in each group.

[0060] (2) siDirect v2.1 tool was used to design siRNA sequences targeting the PDE11A gene, where: Primer design for negative control group: siNC-F: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO. 12) siNC-R: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 13) Primer design for experimental group: siPDE11A-F: 5'-CCGCAUCAUGACACAUACA-3' (SEQ ID NO. 14) siPDE11A-R: 5'-UGUAUGUGUCAUGAUGCGG-3' (SEQ ID NO. 15) The above four primers (SEQ ID NO. 10-13) were delivered to Shanghai Sangon Biotechnology Technology Service Co., Ltd. for primer sequence synthesis.

[0061] (3) The primers synthesized from the above SEQ ID NO. 12 to 15 were centrifuged and dissolved in 1.5× PBS, and a blank control group and an experimental group were set up respectively.

[0062] Blank control group (PBS): each L. vannamei shrimp was injected with 20 μL of 1.5×PBS; Negative control group (siNC): each L. vannamei shrimp was injected with siNC-F and siNC-R dissolved in 1×PBS, with an injection dose of 2 μg / g / shrimp for siNC-F and siNC-R (i.e., each L. vannamei shrimp was injected with 2 μg per gram of body weight, and so on, the injection dose was increased according to the body weight of each shrimp); Experimental group (siPDE11A): Each shrimp was injected with siPDE11A-F and siPDE11A-R dissolved in 1.5×PBS, with an injection dose of 2 μg / g / shrimp for siPDE11A-F and siPDE11A-R respectively (the same as the negative control group); the injection was once every 4 days, and the injection was targeted at the middle of the second to third abdominal segments. The experiment lasted for 22 days. After the experiment, the hepatopancreas and muscle samples of all shrimp were collected and stored in a -80℃ refrigerator, and the body length of each shrimp was measured (accurate to mm) before each injection and sample collection.

[0063] The measurement results are as follows Figure 8 As shown in the figure, the body length growth of Penaeus 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 growth of Penaeus vannamei in the experimental group was about 1 cm, while the body length growth of the blank control group (PBS) and the negative control group (siNC) was mainly above 1 cm, and the body length growth of Penaeus vannamei in the blank control group (PBS) was the largest, which was 1.5 cm.

[0064] S3. Determination of relative expression level of PDE11A gene in Litopenaeus vannamei (1) Extraction of total RNA from Litopenaeus vannamei Total RNA was extracted from the hepatopancreas and muscle of the above-collected Litopenaeus vannamei using 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 steps of the RNA extraction experiment using the Trizol method are as follows: 1) Wash the pipette tips, eight-bar, grinding magnetic beads, scissors, tweezers and other experimental equipment, put them in the autoclave for sterilization, and then put them in the oven for use; 2) Cut 50 μg of Penaeus vannamei tissue (hepatopancreas or muscle tissue) and put it into a 1.5 ml enzyme-free centrifuge tube, and soak the tissue in 500 μL Trizol; 3) Add 3-5 grinding magnetic beads to the centrifuge tube and place the centrifuge tube in a tissue grinder for grinding at 70 Hz and 4°C for 3 min. 4) After grinding the tissue, add 500 μL Trizol to the centrifuge tube, mix well and place on ice for 5 min; 5) Centrifuge at 12000 rpm, 4°C for 5 min; 6) Take the supernatant and transfer it to a new centrifuge tube. Add about 200 μL of chloroform, shake vigorously for about 15 seconds to mix, and place on ice for 15 minutes. 7) Centrifuge at 12000 rpm, 4°C for 15 min; 8) Aspirate the supernatant into another centrifuge tube, add an equal amount of -20℃ precooled isopropanol, mix gently, and place at -20℃ for 2 h; 9) Centrifuge at 1200 rpm, 4°C for 10 min; 10) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, shake gently to suspend the precipitate, and centrifuge at 10,000 rpm, 4°C for 5 min; 11) Discard the supernatant, add 1 mL of pre-cooled anhydrous ethanol, shake gently to suspend the precipitate, and centrifuge at 10,000 rpm, 4°C for 5 min; 12) Discard the supernatant, open the centrifuge tube cover and air dry it in a clean bench for 5-10 minutes; 13) Add about 50 μL of DEPC water to dissolve the precipitate; 14) Take 1 µL RNA sample and measure its concentration and purity using Nanodrop 2000 micro-volume spectrophotometer; 15) Take 1 µg RNA sample and use 1.5% agarose gel electrophoresis to check its integrity and observe whether there are obvious 28S and 18S rRNA bands; 16) Store the qualified RNA samples in a -80℃ refrigerator.

[0065] Among them, the results of total RNA extraction from hepatopancreas after RNA interference are as follows Fig. 9 As shown in a, M is a marker, 1-12 are total RNA of hepatopancreas after RNA interference, and there is a brighter band at about 500 bp of 28S; The results of total RNA extraction from muscle are as follows Fig. 9 As shown in b, M is a marker, 1-12 is the total muscle RNA after RNA interference, and there is a bright band around 500 bp of 28S, indicating that the quality of total RNA extraction is good and can be used for subsequent experiments.

[0066] (2) Reverse transcription The total RNA extracted from Penaeus vannamei was used as a template and reverse transcription was performed using the All-In-One 5X RT MasterMix kit (the following reagents are included in the kit). The reverse transcription system was: 5X All-In-One RTMasterMix 4 μL, total RNA 1 μg, and the volume was adjusted to 20 μL using RNase-free water; the reverse transcription program was: 37℃ 15 min, 60℃ 10 min, 95℃ 3 min, and stored at 4℃ to obtain the cDNA of Penaeus vannamei.

[0067] (3) qRT-PCR The cDNA of Penaeus vannamei obtained above was diluted 5 times as a template, 18S rRNA was used as an internal reference gene, and specific primers for PDE11A gene and internal reference gene were designed through NCBI, wherein: Upstream primer 18S-F: 5′-TATACGCTAGTGGAGCTGGAA-3′ (SEQ ID NO. 16); Downstream primer 18S-R: 5′-GGGGAGGTAGTGACGAAAAAT-3′ (SEQ ID NO. 17); Upstream primer PDE11A-F: 5′-ATGCTGGACCTTGCACGAAT-3′ (SEQ ID NO. 18); Downstream primer PDE11A-R: 5′-TTGATGGGGAAGCGTGACTC-3′ (SEQ ID NO. 19).

[0068] The BlasTaq 2X qPCR MasterMix kit was used to perform qRT-PCR to detect the expression level of the target gene. The reaction system of qRT-PCR was: 2 μL cDNA of Penaeus vannamei, 10 μL BlasTaq 2X qPCR MasterMix, 0.5 μL upstream primer (10 μM); 0.5 μL downstream primer (10 μM), 7 μL DEPC water.

[0069] Each sample was set up with 3 biological replicates, and each biological replicate was set up with 4 technical replicates. The samples were added to the 96-well plate, centrifuged at 3000 rpm for 1 min, and qRT-PCR was performed using the fluorescent quantitative gene amplification instrument qTOWER3G. The reaction procedure of qRT-PCR was as follows: Pre-denaturation at 95℃ for 30 s, Denaturation at 95°C for 10 s, Annealing and extension were performed at 60°C for 30 s, with a total of 40 cycles from denaturation to annealing and extension.

[0070] The specificity of the primers was verified by melting curve analysis using 2- △△t The relative expression level of the target gene was calculated.

[0071] qRT-PCR results Fig.10 As shown, the relative expression of PDE11A gene in the experimental group was lower than that in the control group (blank control group and negative control group) in both the hepatopancreas and muscle tissue of Penaeus vannamei. In particular, in the hepatopancreas of Penaeus vannamei, the relative expression of PDE11A gene in the experimental group was significantly lower than that in the control group; in the muscle of Penaeus vannamei, the relative expression of PDE11A gene in the experimental group was extremely significantly lower than that in the control group. The results showed that siPDE11A effectively inhibited the expression of PDE11A gene and had a significant effect on the growth traits of Penaeus vannamei.

[0072] In summary, the PDE11A gene of Penaeus vannamei can regulate the growth rate of Penaeus vannamei. The larval stage of Penaeus vannamei was briefly treated with RNA interference technology, and the results showed that siPDE11A significantly and extremely significantly inhibited the expression of the PDE11A gene in the hepatopancreas and muscle tissue, respectively, reducing the expression waste of the PDE11A gene of Penaeus vannamei in the larval stage, and narrowing the growth rate differences of Penaeus vannamei individuals in the group in a short time; after restoring the expression of the PDE11A gene of Penaeus vannamei, the overexpression of the PDE11A gene can be used to accelerate the growth of Penaeus vannamei individuals, improve the breeding efficiency of Penaeus vannamei, and increase the economic value.

[0073] Comparative Example 1 The difference between this comparative example and Example 1 is that the primers used are: Trmt10a-F: 5'-CGGTGTTCATAAAGAGGATAC-3' (SEQ ID NO. 20); Trmt10a-R: 5'-CTCTCCACTTTTGTGTCGTTC-3' (SEQ ID NO. 21).

[0074] The primers used in this comparative example are primers for detecting the SNP sites of the rapid growth rate of Penaeus vannamei that are publicly reported in literature.

[0075] The results show that the primers provided in this comparative example 1 cannot detect the SNP molecular marker combination in Example 1, and the PCR reaction using the primer set provided by the present invention can quickly detect the SNP molecular marker combination in Example 1.

[0076] It can be concluded that the present invention located two SNP molecular marker sites associated with body length on the PDE11A gene of Penaeus vannamei through whole genome 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; 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 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. The AA / AA genotype in this SNP molecular marker combination is used to select parents, which can increase the growth rate of the breeding population, provide innovative tools for the molecular breeding of Penaeus vannamei, shorten the breeding cycle, and help industry development and improve efficiency.

[0077] 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 to exclude any equivalent features of the illustrations and descriptions (or parts thereof), and it should be recognized that various modifications that may exist should also be included in the scope of the claims. Other modifications, changes and substitutions may also exist. Accordingly, the claims should be deemed to cover all such equivalents.

[0078] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions may be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A SNP molecular marker combination, characterized in that: The sequence of the SNP molecular marker combination is 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.

2. Use of the SNP molecular marker combination as claimed in claim 1 in breeding of Penaeus vannamei.

3. The use of the SNP molecular marker combination according to claim 2 in the breeding of Penaeus vannamei, characterized in that: The vannamei shrimp breeding includes body length screening, parent shrimp individual identification, group breeding or strain maintenance of vannamei shrimp.

4. A primer set, characterized in that: The primer set comprises one, a pair or more of the following base sequences: PDE11A-2877-Fw: 5'-AGTGACTCGTAGCGAAAGCC-3'; PDE11A-2877-Rw: 5'-CCTGTGTATGCCACGCTCTT-3'; PDE11A-8932-Fw: 5'-TGCCTTGAAATGACGTGTGTG-3'; PDE11A-8932-Rw: 5'-ACCACTGGGTAGTCTGAAATG-3'.

5. Use of the primer set as claimed in claim 4 in breeding of Penaeus vannamei.

6. A detection kit comprising the primer set according to claim 4.

7. Use of the detection kit as claimed in claim 6 in breeding of Penaeus vannamei.

8. A method for breeding Litopenaeus vannamei using the SNP molecular marker combination of claim 1, 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; 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.

9. The method for breeding Penaeus vannamei according to claim 8, 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; the genotype of the SNP combination site is A / G at position 2877 and A / G at position 8932 of the sequence shown in SEQ ID NO.

1.

10. The method for breeding Penaeus vannamei according to claim 8, 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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