SNP (Single Nucleotide Polymorphism) molecular marker associated with body length growth traits of litopenaeus vannamei, detection primer and application of SNP molecular marker

Abstract

The invention discloses a litopenaeus vannamei body length growth trait associated SNP molecular marker, a detection primer and application thereof. The sequence of the SNP molecular marker is as shown in SEQ ID NO.1, the SNP site is located at the 282 site from the 5'end of the sequence as shown in SEQ ID NO.1, and the polymorphism of the SNP molecular marker is T / C type. The SNP locus can be used for body length screening of litopenaeus vannamei in litopenaeus vannamei assisted breeding, and a primer designed according to the sequence of SEQ ID NO.1 or a kit prepared from the primer can be used for detecting the SNP locus. The SNP molecular marker Chr3711743678 provided by the invention can be used for assisting in selecting rapid growth parents, and the CC genotype in the SNP molecular marker is used for selecting the parents, so that the growth speed of breeding populations can be increased, an innovative tool is provided for molecular breeding of litopenaeus vannamei, the breeding period is shortened, and industrial development and efficiency improvement are 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 SNP molecular marker associated with the body length growth trait of 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] Therefore, the development of SNP molecular markers for the body length growth traits of Penaeus vannamei is of great significance for the 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

[0006] The purpose of the present invention is to provide a SNP molecular marker and detection primer associated with the body length growth trait of 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: On one hand, the present invention provides a SNP molecular marker, the sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type.

[0007] Another aspect of the present invention provides an application of a SNP molecular marker in assisted breeding of Penaeus vannamei, wherein the sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; and the polymorphism of the SNP molecular marker is T / C type.

[0008] Furthermore, assisted breeding is used to screen the body length of Penaeus vannamei.

[0009] Another aspect of the present invention provides a primer pair, the base sequence of the primer pair is: Forward primer Fw: 5′-TGTAGAAATGCGCTCACATGC-3′; Reverse primer Rw: 5′-ATTACAGACGCACAGCAGCA-3′; Furthermore, the primer pair is used to detect the SNP molecular marker, the sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type.

[0010] Another aspect of the present invention provides an application of a primer pair in assisted breeding of Penaeus vannamei.

[0011] Another aspect of the present invention provides a detection kit for Penaeus vannamei, the detection kit comprising a primer pair; the base sequence of the primer pair is: Forward primer Fw: 5′-TGTAGAAATGCGCTCACATGC-3′; Reverse primer Rw: 5′-ATTACAGACGCACAGCAGCA-3′.

[0012] Another aspect of the present invention provides the use of the detection kit in assisted breeding of Penaeus vannamei.

[0013] Another aspect of the present invention provides a method for assisted breeding of Penaeus vannamei, comprising the following steps: Step A: detecting the SNP molecular marker of Litopenaeus vannamei to obtain the genotype of the SNP site; the sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type; Step B: screening according to the genotype of the SNP site; the genotype of the SNP site is CC, TC or TT genotype; the screening is based on the body length of the vannamei shrimp; Step C: breeding the shrimp Penaeus vannamei with the CC genotype of the SNP locus.

[0014] 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, PCR amplification was performed using the following primer pair: forward primer Fw: 5'-TGTAGAAATGCGCTCACATGC-3', reverse primer Rw: 5'-ATTACAGACGCACAGCAGCA-3'; The PCR amplification system was a 25 μL amplification system; the 25 μL amplification system included: genomic DNA of Penaeus vannamei (100 ng / μL), 1 μL; forward primer Fw (10 μM), 1 μL; reverse primer Rw (10 μM), 1 μL; 2×Taq PlusMaster Mix Ⅱ (Plus), 12.5 μL; enzyme-free sterile water, 9.5 μL; The PCR amplification procedure included: initial denaturation at 95°C for 3 min; then 32 cycles of: 95°C for 15 s; 56°C for 20 s; 72°C for 40 s; full extension at 72°C for 10 min; The PCR amplification product is obtained, and the PCR amplification product is sequenced to obtain the genotype of the SNP site; the genotype of the SNP site is C / T at position 282 of the sequence shown in SEQ ID NO.1.

[0015] Furthermore, the body length of Penaeus vannamei individuals with the CC genotype of the SNP site>the body length of Penaeus vannamei individuals with the TC genotype of the SNP site>the body length of Penaeus vannamei individuals with the TT genotype of the SNP site.

[0016] Furthermore, the SNP site genotype of the Penaeus vannamei CC genotype is retained, and breeding is carried out by pairing and breeding with the Penaeus vannamei parents of the CC genotype.

[0017] The present invention locates the body length-associated SNP molecular marker Chr37_11743678 located on chromosome 37 of Penaeus vannamei through whole genome association analysis. The sequence of the SNP molecular marker is shown in SEQ ID NO.1. The SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is T / C type. Primers SEQ ID NO.2 and SEQ ID NO.3 designed according to the sequence of SEQ ID NO.1 can detect the SNP molecular marker, and the above primers can also be prepared into a kit. Therefore, the SNP molecular marker Chr37_11743678 provided by the present invention can be used to assist in the selection of fast-growing parents. The CC genotype in this SNP molecular marker 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 industrial development and efficiency improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The Manhattan plot of the genome-wide association analysis of the body length of Penaeus vannamei in Example 1; Figure 2 It is a box plot of the body length distribution of individuals of different genotypes of Penaeus vannamei in Example 1 (wherein the abscissa is genotype, red is CC genotype, green is CT genotype, and blue is TT genotype; the ordinate is the body length of Penaeus vannamei, in (mm); "*" represents the P value, and there is a significant difference in the individual body length of Penaeus vannamei between the two genotypes; "****" represents that there is a very significant difference in the individual body length of Penaeus vannamei between the two genotypes); Figure 3 It is the agarose gel electrophoresis diagram of the genomic DNA of Penaeus vannamei in Example 2 (wherein A is the electrophoresis result of the genomic DNA of Penaeus vannamei numbered 1-8; B is the electrophoresis result of the genomic DNA of Penaeus vannamei numbered 9-16; M is a marker; 1-16 are all target genes, all of which are more than 5000 bp in length and have good integrity); Figure 4It is an agarose gel electrophoresis diagram of the PCR amplification product of Penaeus vannamei in Example 2 (wherein, A is the electrophoresis result of the genomic DNA of Penaeus vannamei numbered 1-8; B is the electrophoresis result of the genomic DNA of Penaeus vannamei numbered 9-16; M is a marker; 0 is a blank control: enzyme-free sterile water; 1-8 and 9-16 are both target sequence PCR products, with a length of about 348 bp); Figure 5 is a flowchart illustrating the sequence sequencing in Example 2; Figure 6 This is the peak diagram of the sequencing results of the PCR amplification product of Penaeus vannamei in Example 2 (wherein the 219th genotype is the target genotype). DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] 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.

[0023] (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).

[0024] (2) Sources of reagents and consumables Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0025] (3) Source of instruments and equipment Table 2 Sources and models of instruments and equipment required for the experiment

[0026] Example 1

[0027] A SNP molecular marker 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: 996 individuals were randomly selected from the 8-month-old Penaeus vannamei population, and the body length of each Penaeus vannamei 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 sites were obtained. After quality control of the above 14,276,173 SNP sites, a total of 2,706,353 SNP sites were screened.

[0028] S2. Analysis of the correlation between body length and genotype of Penaeus vannamei. The specific reference steps are as follows: (1) Based on the gene resequencing results in step S1, the whole genome SNP genotyping data of the 2,706,353 SNP sites that had been screened were used to perform principal component analysis (PCA) and population structure analysis using PLINK software. The first ten 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 genome-wide association studies (GWAS).

[0029] 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.

[0030] 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).

[0031] 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 molecular marker Chr37_11743678 associated with the body length growth trait of Penaeus vannamei was located, located at position 11743678 on chromosome 37 of Penaeus vannamei, and the nucleotide sequence of the SNP molecular marker Chr37_11743678 is shown in SEQ ID NO.1. Through genotyping, it was found that the genotype of the SNP molecular marker Chr37_11743678 was CC, TC and TT, wherein the SNP molecular marker, as shown in SEQ ID NO.1, has a T or C base at the 282nd position from the 5' end, which is a T / C type SNP molecular marker. After the individuals of Penaeus vannamei were classified by genotype, the body length of Penaeus vannamei was analyzed for association.

[0032] (2) One-way analysis of variance (ANOVA) was used to detect the differences in body length among the three genotypes of Litopenaeus vannamei. T-test was used to compare the body lengths of different genotypes, i.e., CC vs TC, CC vs TT, and TC vs TT (Table 3). The body length distribution of individuals of different genotypes was visually displayed using box plots ( Figure 2 ).

[0033] As shown in Table 3, the three genotypes of SNP site Chr37_11743678 all significantly affected the body length growth traits of Penaeus vannamei (one-way ANOVA P.value (P value) < 0.05 was considered to be significantly different; two-way T test P.value (P value) < 0.05 was considered to be significantly different). Among them, the average body length of CC-type Penaeus vannamei individuals was significantly greater than that of TC-type Penaeus vannamei individuals and TT-type Penaeus vannamei individuals (P < 0.05), and the average body length of TC-type Penaeus vannamei individuals was significantly greater than that of TT-type individuals (P < 0.05).

[0034] like Figure 2 As shown in the figure, the box plot shows the distribution of the three genotypes (CC, CT, TT) of the SNP locus lg37_11743678 in the body length trait of Penaeus vannamei. The mean values ​​of each genotype were 211.28 mm (CC), 205.16 mm (CT), and 199.31 mm (TT); the maximum values ​​were 237.63 mm (CC), 246.51 mm (CT), and 250.32 mm (TT); and the minimum values ​​were 188.47 mm (CC), 174.48 mm (CT), and 157.2 mm (TT). The standard deviation of the CT genotype was 12.58, the TT genotype was 11.57, and the CC genotype was 12.47.

[0035] It can be seen that the average body length of individuals with CC genotype is the highest and the distribution is relatively balanced, showing superior growth traits. The significance of body length differences between genotypes was analyzed by pairwise T test. "*" indicates P value < 0.05 (significant difference), and "****" indicates P value < 0.0001 (extremely significant difference). The test results show that the difference between CC and CT genotypes is significant (*), while the difference between CC and TT, CT and TT genotypes is extremely significant (****).

[0036] From this, we can know that the C allele contributes to the growth of the body length of Penaeus vannamei and is a favorable allele, while the T allele has a negative effect on growth. Therefore, the CC type is the best genotype and has important selection value in the breeding process.

[0037] Body length is an important indicator to measure the growth and development of Penaeus vannamei, and is closely related to the individual's adaptability to the environment, economic value, production performance and reproductive capacity.

[0038] During the breeding process, by gradually eliminating individuals carrying unfavorable genotypes (such as TC and TT types) and preferentially retaining individuals with favorable genotypes (such as CC type), the frequency of favorable genotypes at this site can be increased generation by generation, thereby improving the growth performance and market competitiveness of the entire population.

[0039] Table 3 Correlation analysis between molecular marker SNP site Chr37_11743678C / T and body length of Penaeus vannamei

[0040] Example 2

[0041] An application of a SNP molecular marker associated with the body length growth trait of Penaeus vannamei in breeding comprises the following steps: S1. Extraction of genomic DNA from Litopenaeus vannamei 16 shrimps of different sizes were randomly selected from a group of 8-month-old Penaeus vannamei. The antennae of the shrimps 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 shrimps. 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.

[0042] (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.

[0043] (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.

[0044] (4) Purification by adsorption column: Transfer the mixed solution to the CB3 adsorption column, place it in a collection tube, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Then add 500 μL GD buffer and 600 μL PW rinse solution, centrifuge for 30 seconds each time, and wash the adsorption column to remove impurities.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] 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, and the remaining Penaeus vannamei genomic DNA was stored in a -20℃ refrigerator for later use.

[0049] The results of the first agarose gel electrophoresis were as follows Figure 3 As shown, M is a marker, 1-16 are target genes, all of which are longer than 5000 bp and have good integrity.

[0050] S2. Primer design The primer design function module Primer-BLAST of NCBI was used to design primers for the SEQ ID NO.1 sequence (referred to as the target sequence) of the SNP molecular marker of Litopenaeus vannamei: Forward primer Fw: SEQ ID NO.2: 5'-TGTAGAAATGCGCTCACATGC-3' Reverse primer Rw: SEQ ID NO. 3: 5′-ATTACAGACGCACAGCAGCA-3′.

[0051] The synthesized SNP molecular marker primers (Fw and Rw) were sent to Beijing Qingke Biotechnology Co., Ltd. for primer synthesis and used in subsequent PCR amplification reactions.

[0052] S3. PCR amplification reaction Using the genomic DNA of Penaeus vannamei obtained in step S1 of Example 2 as a template, the SNP molecular marker primers (SEQ ID NO.2 and SEQ ID NO.3) synthesized in step S2 of Example 2 were used to perform PCR amplification reaction on the target sequence. 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.

[0053] (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℃.

[0054] After the PCR amplification reaction was completed, the PCR amplification product was obtained and detected by 1.5% agarose gel electrophoresis.

[0055] The results of the second agarose gel electrophoresis were as follows Figure 4 As shown, M is Marker; 1 and 10 are blank controls: enzyme-free sterile water; 2-9 and 11-18 are target sequences, and the target sequence has a brighter band around 348 bp.

[0056] S4. Verification of SNP sites The PCR amplification product obtained in step S3 was sent to Beijing Novogene Technology Co., Ltd. for forward sequencing. The sequencing primer T was: 5'-CAAAGGTCATCCTCATGTTCAT-3' (SEQ ID NO. 4). For specific primer design and sequencing procedures, see Figure 5 ,Depend on Figure 5 It can be seen that the target sequence SEQ ID NO.1 was PCR amplified by forward primer SEQ ID NO.2 and reverse primer SEQ ID NO.3 to obtain a PCR amplification product, and the PCR amplification product was forward sequenced by sequencing primer SEQ ID NO.4 to obtain the sequencing result SEQ ID NO.5 of the SNP site Chr37_11743678.

[0057] According to the sequencing peak diagram of PCR amplification products ( Figure 6 ) Genotyping was performed on the SNP site Chr37_11743678 (G: SEQ ID NO.5). The sequence length of SEQ ID NO.5 was shorter than that of SEQ ID NO.1, that is, the Chr37_11743678 molecular marker was located at the 282nd base from the 5' end as shown in SEQ ID NO.1, which was T or C, and was a T / C type SNP molecular marker, which was equivalent to the Chr37_11743678 molecular marker was located at the 219th base from the 5' end as shown in SEQ ID NO.5, which was T or C, and was a T / C type SNP molecular marker.

[0058] If there is only one C base peak at position 219, the genotyping result is CC type; if there is only one T base peak at position 219, the genotyping result is TT type; if there are both C base peaks and T base peaks at position 219, the genotyping result is T / C type.

[0059] The body lengths of the tested genotypes of Penaeus vannamei and their genotype frequencies in large and small shrimps are shown in Table 4. The average body length of Penaeus vannamei with genotype CC at position 219 is 226.73 mm, and its genotype frequency in large shrimps is 45%, while its genotype frequency in small shrimps is 0; the average body length of Penaeus vannamei with genotype TC at position 219 is 204.55 mm, and its genotype frequency in large shrimps is 45%, while its genotype frequency in small shrimps is 30%; the average body length of Penaeus vannamei with genotype TT at position 219 is 177.23 mm, and its genotype frequency in large shrimps is 10%, while its genotype frequency in small shrimps is 70%;

[0060] Table 4 Body length corresponding to the genotypes of Litopenaeus vannamei and the genotype frequency distribution in large and small shrimps

[0061] Therefore, the CC genotype is associated with a faster growth rate, while the TT genotype is associated with a slower growth rate. Therefore, by selecting parents for the CC genotype in the SNP site Chr37_11743678 marker, the growth rate of the breeding population can be increased.

[0062] It can be concluded that the present invention locates the body length-associated SNP molecular marker Chr37_11743678 located on chromosome 37 of Penaeus vannamei through whole genome association analysis. The sequence of the SNP molecular marker is shown in SEQ ID NO.1. The SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is T / C type. The primers SEQ ID NO.2 and SEQ ID NO.3 designed according to the SEQ ID NO.1 sequence can detect the SNP molecular marker, and the above primers can also be prepared into a kit. Therefore, the SNP molecular marker Chr37_11743678 provided by the present invention can be used to assist in the selection of fast-growing parents. The CC genotype in this SNP molecular marker 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 efficiency improvement.

[0063] 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.

[0064] 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, characterized in that: The sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type.

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

3. The use of the SNP molecular marker according to claim 1 in assisted breeding of Penaeus vannamei, characterized in that: The auxiliary breeding is the body length screening of Penaeus vannamei.

4. A primer pair, characterized in that: The base sequence of the primer pair is: Forward primer Fw: 5′-TGTAGAAATGCGCTCACATGC-3′; Reverse primer Rw: 5′-ATTACAGACGCACAGCAGCA-3′.

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

6. A detection kit for Penaeus vannamei, characterized in that: The kit includes a primer pair; the base sequence of the primer pair is: Forward primer Fw: 5′-TGTAGAAATGCGCTCACATGC-3′; Reverse primer Rw: 5′-ATTACAGACGCACAGCAGCA-3′.

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

8. A method for assisted breeding of Penaeus vannamei, characterized in that: The following steps are involved: Step A: detecting the SNP molecular marker of Litopenaeus vannamei to obtain the genotype of the SNP site; the sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at the 282nd position from the 5' end of the sequence shown in SEQ ID NO.1; the polymorphism of the SNP molecular marker is T / C type; Step B: screening according to the genotype of the SNP site; the genotype of the SNP site is CC, TC or TT; the screening is based on the body length of the vannamei shrimp; Step C: breeding the shrimp Penaeus vannamei with the CC genotype of the SNP site.

9. The method for assisted breeding of 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 pair: forward primer Fw: 5'-TGTAGAAATGCGCTCACATGC-3', reverse primer Rw: 5'-ATTACAGACGCACAGCAGCA-3'; The PCR amplification system is a 25 μL amplification system; the 25 μL amplification system includes: 1 μL of genomic DNA of Penaeus vannamei with a concentration of 100 ng / μL; 1 μL of forward primer Fw with a concentration of 10 μM; 1 μL of reverse primer Rw with a concentration of 10 μM; 12.5 μL of 2×Taq Plus Master Mix Ⅱ Plus; 9.5 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°C for 20 s; 72°C for 40 s; full extension at 72°C for 10 min; A PCR amplification product is obtained, and the PCR amplification product is used for sequencing to obtain the genotype of the SNP site; the genotype of the SNP site is C / T at position 282 of the sequence shown in SEQ ID NO.

1.

10. The method for assisted breeding of Penaeus vannamei according to claim 8, characterized in that: The basis for screening according to the genotype of the SNP site is: the body length of the vannamei shrimp individuals with the CC genotype of the SNP site> the body length of the vannamei shrimp individuals with the TC genotype of the SNP site> the body length of the vannamei shrimp individuals with the TT genotype of the SNP site.

11. The method for assisted breeding of Penaeus vannamei according to claim 8, characterized in that: The vannamei shrimp with CC genotype at the SNP site is retained, and breeding is performed by pairing and breeding the vannamei shrimp parents with CC genotype.

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