Use of SNP site combination of litopenaeus vannamei in trait evaluation of mixed-family culture, and probe and kit

Abstract

The use of an SNP site combination of Litopenaeus vannamei in trait evaluation of mixed-family culture, a probe and a kit. The SNP site combination of Litopenaeus vannamei comprises 1125 SNP sites, wherein the physical positions are determined on the basis of alignment with a reference genome sequence of Litopenaeus vannamei. Also disclosed are a molecular probe for capturing the SNP site combination and a kit comprising the molecular probe. By means of using the SNP site combination for paternity identification, the present invention can achieve the mixed culture of individuals of different families in the early stage, so that the common environmental effect caused by separate family culture in the early stage is effectively reduced, and the accuracy of a trait evaluation result of mixed culture is improved; moreover, the genetic relationship between individuals can be accurately calculated. Compared with physical marks, the present invention avoids the phenomena of falling and dislocation of marks on individuals and human identification errors, thus obtaining accurate pedigrees.

Description

Application of SNP site combination of Litopenaeus vannamei in evaluation of mixed breeding traits of its family, and probe and kit TECHNICAL FIELD

[0001] The present application relates to the technical field of Litopenaeus vannamei breeding, and particularly to application of SNP site combination of Litopenaeus vannamei in evaluation of mixed breeding traits of its family, and a probe and a kit. BACKGROUND

[0002] Litopenaeus vannamei is the highest yield of shrimp species in the world at present, which has the characteristics of fast growth, strong disease resistance and significant economic benefit of breeding. Litopenaeus vannamei was introduced into China in 1988, and rapidly spread across the country after solving the problem of large-scale breeding around 2000. After more than 30 years of development, the breeding technology of Litopenaeus vannamei has gradually matured, and the yield has increased year by year, reaching more than 6.3 million tons in 2021. Excellent seed shrimp is the source of high-quality development of the industry, and large-scale family selection technology is the core technology to support the selection of Litopenaeus vannamei seed shrimp.

[0003] In the traditional large-scale family selection technology, the performance of growth, survival and other traits of different families needs to be evaluated by mixed breeding. In order to distinguish different families, physical markers are mainly used to identify families, for example: different color combinations of visible implant elastomer (VIE) are used to identify different family individuals for pedigree tracking. In this process, shrimp family individuals must be bred separately for 50-70 days until the juvenile shrimp grow to a large enough size to allow physical marking. The longer the family is bred separately, the greater the environmental difference between families, resulting in a common environmental effect that affects the accuracy of mixed breeding trait evaluation. In addition, when using physical markers, individual markers may fall off, misposition, and human recognition errors may occur, which will all lead to pedigree errors and further affect the accuracy of mixed breeding trait evaluation.

[0004] SUMMARY TECHNICAL PROBLEM

[0005] The purpose of the present application is to provide an application of SNP site combination of Litopenaeus vannamei in evaluation of mixed breeding traits of its family, and a probe and a kit, which aims to solve the problem that the common environmental effect caused by separate feeding and the existence of individual marker falling off, misposition and human recognition error in the large-scale family selection technology using physical markers in the prior art affect the accuracy of mixed breeding trait evaluation of Litopenaeus vannamei family. TECHNICAL SOLUTION

[0006] In one aspect, the application provides a SNP site combination of Litopenaeus vannamei for use in evaluating the mixed breeding trait of the Litopenaeus vannamei, wherein the SNP site combination comprises 1125 SNP sites, the physical positions of the SNP sites are determined based on the alignment of the reference genome sequence of the Litopenaeus vannamei, and the information of the SNP sites is shown in Table 1, wherein ID is the site number, Scaffold is the Scaffold name of the reference genome of the Litopenaeus vannamei where the site is located, Position is the physical position of the site on the Scaffold, and Ref is the reference genome base name of the Litopenaeus vannamei of the site.

[0007] Table 1 Information of SNP sites

[0008] The application uses the SNP site combination for parentage identification, and different families do not need to be bred separately in the early stage, thereby realizing the mixed breeding of individuals in different families in the early stage, effectively reducing the common environmental effect caused by separate breeding of families in the early stage, and improving the accuracy of evaluating the mixed breeding trait of the Litopenaeus vannamei. Moreover, the SNP site combination of the application can be used to accurately calculate the genetic relationship between individuals, and does not cause the phenomena of individual marker loss, misplacement and human identification error, thereby obtaining an accurate pedigree. When the mixed breeding trait of the Litopenaeus vannamei is evaluated by using the SNP site combination, the family of the Litopenaeus vannamei can be accurately identified, and the survival trait and the growth trait of the mixed breeding of the Litopenaeus vannamei can be effectively evaluated. The SNP (Single Nucleotide Polymorphism) molecular marker has the characteristics of large quantity, wide distribution on the genome, high-throughput identification, etc. ID, i.e. identification, site number; Scaffold, support; Position, physical position; Ref, i.e. reference, reference genome base name; the acquisition website of the reference genome sequence of the Litopenaeus vannamei in the application is: https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / 6689 / .

[0009] The molecular probe applied in the evaluation of the mixed breeding traits of Litopenaeus vannamei family is a DNA single-strand probe, and the molecular probe is 1-4 nucleotide sequences with differences designed according to the SNP site group of Litopenaeus vannamei in the application of the evaluation of the mixed breeding traits of the family, each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP site. DNA (deoxyribonucleic acid) is a deoxyribonucleotide, and bp is a base pair.

[0010] The molecular probe of the application is a molecular probe for detecting the SNP site group of Litopenaeus vannamei, and the molecular probe group can perform site-specific capture on 1125 target regions, single-strand nucleotide synthesis is performed according to the above-mentioned designed nucleotide sequence, the synthesis length is 110 bp, the 5' end is modified with a biotin group DNA nucleotide sequence, and the molecular probe; the synthesized molecular probe is mixed in equal molar mass, an EDTA and Tris-HCL mixed solution is used to prepare a probe mixed solution. EDTA, ethylenediaminetetraacetic acid, chemical formula is C 10 H 16 N2O8; Tris-HCL, Tris (hydroxymethyl) Aminomethane Hydrochloride, Tris (hydroxymethyl) Aminomethane Hydrochloride, molecular formula is C4H 12 ClNO3.

[0011] The kit applied in the evaluation of the mixed breeding traits of Litopenaeus vannamei family comprises a probe mixed solution and a hybrid capture reagent, and the probe mixed solution is prepared from the molecular probe applied in the evaluation of the mixed breeding traits of the Litopenaeus vannamei family.

[0012] The kit for application in the evaluation of the mixed culture trait of Litopenaeus vannamei family of the application is composed of independently packaged probe mixture and hybridization capture reagent, the probe mixture is prepared from molecular probes, and the hybridization capture reagent is usually composed of Block I (blocker I), Block II (blocker II), 2xHyb Buffer (2x hybridization buffer), Hyb Buffer Enhancer (hybridization buffer enhancer), Primer Mix (primer mixture), PCR Master Mix (PCR master mix), 2xBeads Wash Buffer (2x bead wash buffer), 10xWash Buffer I (10x wash buffer I), 10xWash Buffer II (10x wash buffer II), 10xWash Buffer III (10x wash buffer III), 10xStrigent Wash Buffer (10x stringent wash buffer), DNA Probe Beads (DNA probe beads), and DNA Clean Beads (DNA clean beads), and the hybridization capture reagent is preferably from Shijiazhuang Boruidi Biotechnology Co., Ltd., the kit has low cost and good accuracy.

[0013] As a preferred embodiment, the application of the kit in genotype information identification comprises the following steps:

[0014] 1) The DNA of the Litopenaeus vannamei to be tested is broken, end-repaired, adapter-ligated, purified to obtain a sequencing library, then the library is amplified and purified to obtain the purified library DNA;

[0015] 2) The probe mixture and hybridization capture reagent in the kit are added to the purified library DNA obtained in step 1) for hybridization capture, elution, removal of non-target DNA fragments, and acquisition of target DNA fragments including the SNP site, and the obtained target DNA is amplified and purified to construct a targeted sequencing library;

[0016] 3) The targeted sequencing library obtained in step 2) is detected, and after passing the detection, the DNA sequencing is performed by using the second-generation sequencing technology, the sequencing results are compared with the Litopenaeus vannamei reference genome sequence, and thus the genotype information of the Litopenaeus vannamei to be tested is obtained.

[0017] The kit for application in the evaluation of the mixed breeding trait of Litopenaeus vannamei family according to the application can be used for identifying the genotype information of Litopenaeus vannamei, is a genotyping method of the Litopenaeus vannamei genome based on targeted capture sequencing, and is a method for obtaining a target DNA fragment by using the molecular probe of the application to capture a target region segment, the target region segment being a DNA fragment including a target site, and then performing DNA sequencing and alignment with a reference genome sequence of Litopenaeus vannamei, and finally obtaining the genotype information of the to-be-tested Litopenaeus vannamei. The genotyping method is simple to operate, low in cost, consistent in site between different sequencing batches, flexible in site, and upgradeable.

[0018] As a preferred embodiment, the application of the kit in the evaluation of the mixed breeding trait of Litopenaeus vannamei family comprises the following steps:

[0019] S1 Preparation

[0020] 1) Selecting juvenile shrimp of different families, the number of families being not less than 10;

[0021] 2) Taking the paternal individual, the maternal individual and the sibling individual of the juvenile shrimp in step 1) as target individuals, and identifying the genotype information of the target individuals by using the steps in the application of the kit in the identification of genotype information;

[0022] S2 Mixed breeding

[0023] 3) Randomly taking 30-50 juvenile shrimps in each family in step 1) and putting them into multiple parallel breeding ponds, and then carrying out mixed breeding, the water temperature being not less than 23℃, and the time being 60-90 days, to obtain test individuals;

[0024] S3 Trait evaluation

[0025] 4) Counting the number of test individuals in each breeding pond, taking the test individuals, and measuring the body weight of the test individuals;

[0026] 5) Identifying the genotype information of the test individuals by using the steps in the application of the kit in the identification of genotype information, the test individuals being obtained in step 4);

[0027] 6) Reconstructing a pedigree by using the genotype information of the test individuals obtained in step 5) and the genotype information of the target individuals obtained in step 2), to obtain the family information to which the test individuals belong;

[0028] 7) According to the family information obtained in step 6), counting the survival number and the body weight of the test individuals in different families, to obtain the survival trait and the growth trait of the mixed breeding of different Litopenaeus vannamei families.

[0029] The kit for evaluating the mixed breeding traits of Litopenaeus vannamei families in the application can be used for evaluating the mixed breeding traits of Litopenaeus vannamei families, and is a method for evaluating the mixed breeding traits of Litopenaeus vannamei families, specifically, a method for evaluating the survival traits and growth traits of the mixed breeding of Litopenaeus vannamei families, and is a test method for the mixed breeding of Litopenaeus vannamei families, which uses SNP site combinations for parentage identification, and different families do not need to be bred separately, thereby replacing the traditional VIE marker, realizing the mixed breeding of individuals in different families at an early stage, effectively reducing the common environmental effects caused by single-family breeding at an early stage, and improving the accuracy of the evaluation of the mixed breeding traits of Litopenaeus vannamei families; moreover, compared with the traditional VIE marker for identifying families, the SNP site combinations in the application can accurately calculate the genetic relationship between individuals, and the phenomena of individual marker loss, misplacement and human identification errors do not occur, so that an accurate pedigree is obtained. The method for evaluating the mixed breeding traits of Litopenaeus vannamei families in the application has low cost, and effectively evaluates the survival traits and growth traits of the mixed breeding of Litopenaeus vannamei families.

[0030] As a preferred embodiment, in step 1), the larvae are in the P15-P40 stage. The larvae in the P15-P40 stage are selected for mixed breeding test in the application, and P15-P40 refers to 15 days to 40 days after metamorphosis of the prawn into larvae, and the larvae are 15-40 days old and have good growth; the mixed breeding time is generally 60-90 days in the application, at this time, the body weight of the test individual is close to 15 g; in addition, the breeding environment and survival rate are also considered to determine the mixed breeding time.

[0031] As a preferred embodiment, in step 2), the sibling individuals are in the P45 stage or later. The sibling individuals in the P45 stage or later are selected for genotype identification of the larvae in the application, and P45 refers to 45 days after metamorphosis of the prawn into larvae, and P45 or later refers to more than 45 days after metamorphosis of the prawn into larvae, and such sibling individuals are large and DNA extraction is easy, thereby fully ensuring the quality of genotype identification.

[0032] As a preferred embodiment, in step 2), in the process of identifying the genotype information of the target individual: if the parents of the family are successfully genotyped, 1-3 sibling individuals in the family are selected for genotyping, so as to obtain the genotype information of the target individual; if one of the parents of the family is not successfully genotyped, 2-4 sibling individuals in the family are selected for genotyping, so as to obtain the genotype information of the target individual; if both of the parents of the family are not successfully genotyped, 3-5 sibling individuals in the family are selected for genotyping, so as to obtain the genotype information of the target individual. Different genotyping strategies are adopted for the target individual in the application, so that the number of target individuals is sufficient, thereby meeting the needs of subsequent identification of the family information of the test individual.

[0033] As a preferred embodiment, the culture density of each family of juvenile shrimp in each culture pond in step 4) is 100-300 tails / m 3 Generally, 2-3 culture ponds are arranged to implement parallel mixed culture, and the number of tails of the individual juvenile shrimp in each family is adjusted according to the culture density and the number of families.

[0034] As a preferred embodiment, in the case that the genotype information of the father and the mother in the target individual exists, the genotype information of the father and the mother in the test individual and the target individual is used to generate a pedigree to obtain the family information to which the test individual belongs; in the case that the genotype information of the father and the mother in the target individual does not exist, clustering analysis is performed by using the genotype information of the sibling individual in the test individual and the target individual, and the family information to which the test individual belongs is obtained according to the known family information of the sibling individual in the target individual. When determining the family information to which the test individual belongs, the present application considers comprehensively, can accurately calculate the kinship between individuals, and can accurately distinguish the families of Litopenaeus vannamei. Advantages

[0035] Compared with the prior art, the present application has the advantages that: the present application uses SNP site combination for parentage identification, different families do not need to be cultured separately, replaces the traditional VIE marker, realizes the mixed culture of individuals in different families at an early stage, effectively reduces the common environmental effect caused by single-family culture at an early stage, and improves the accuracy of the evaluation of the mixed culture traits of Litopenaeus vannamei families; moreover, compared with the traditional VIE marker for identifying families, the SNP site combination of the present application can accurately calculate the kinship between individuals, can accurately distinguish the families of Litopenaeus vannamei, and does not have the phenomena of individual marker falling off, misplacement and human identification error, so that an accurate pedigree is obtained. The method for evaluating the mixed culture traits of Litopenaeus vannamei families of the present application has low cost, and realizes effective evaluation of the survival traits and growth traits of the mixed culture of Litopenaeus vannamei families. BRIEF DESCRIPTION OF DRAWINGS

[0036] Fig. 1 is a flow chart of the method for evaluating the mixed culture traits of Litopenaeus vannamei families provided by the present application;

[0037] Fig. 2 is a column chart of the survival rate of the test individual obtained in Example 2 of the present application;

[0038] Fig. 3 is a box chart of the body weight of the test individual obtained in Example 2 of the present application;

[0039] In Fig. 3, the black points represent the harvest body weight of the individual, and the box represents the quartile range of the harvest body weight data of the individual;

[0040] In the box, the lower line represents the lower quartile (the first quartile, Q1);

[0041] The upper line represents the upper quartile (the third quartile, Q3);

[0042] The middle line represents the median (Q2);

[0043] The length of the box represents the interquartile range (IQR). Best mode of the present application

[0044] The technical solutions of the present application will be described clearly and completely below in combination with specific embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0045] The application of a SNP site combination of Litopenaeus vannamei in evaluation of traits of mixed breeding of the Litopenaeus vannamei, the SNP site combination comprises 1125 SNP sites, the physical position of the SNP sites is determined based on sequence alignment of a reference genome of the Litopenaeus vannamei, and information of the SNP sites is shown in Table 1, wherein ID is a site number, Scaffold is a Scaffold name of the reference genome of the Litopenaeus vannamei where the site is located, Position is a physical position of the site on the Scaffold, and Ref is a base name of the reference genome of the Litopenaeus vannamei of the site.

[0046] A molecular probe applied in evaluation of traits of mixed breeding of Litopenaeus vannamei, the molecular probe is a DNA single-strand probe, the molecular probe is designed according to 1-4 nucleotide sequences with differences in the application of the SNP site combination of the Litopenaeus vannamei in evaluation of traits of mixed breeding of the Litopenaeus vannamei, each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP site.

[0047] A kit applied in evaluation of traits of mixed breeding of Litopenaeus vannamei, the kit comprises a probe mixture and a hybridization capture reagent, and the probe mixture is prepared from the molecular probe applied in evaluation of traits of mixed breeding of the Litopenaeus vannamei.

[0048] Preferably, the kit is applied in identification of genotype information, and the application comprises the following steps:

[0049] 1) The DNA of the Litopenaeus vannamei to be tested is broken, end-repaired, adapter-ligated, purified to obtain a sequencing library, then the library is amplified and purified to obtain purified library DNA;

[0050] 2) The purified library DNA obtained in step 1) is added with probe mixture and hybridization capture reagent in the kit, hybridization capture is performed, elution is performed, DNA fragments of non-target regions are removed, target DNA fragments including the SNP site are obtained, the obtained target DNA is amplified and purified to construct a targeted sequencing library;

[0051] 3) The targeted sequencing library obtained in step 2) is detected, after detection, DNA sequencing is performed by using the second-generation sequencing technology, and the sequencing results are aligned on the Litopenaeus vannamei reference genome sequence, so as to obtain the genotype information of the Litopenaeus vannamei to be tested.

[0052] Preferably, the kit is applied in the evaluation of mixed breeding traits of Litopenaeus vannamei family, comprising the following steps:

[0053] S1 Preparation

[0054] 1) Selecting juvenile shrimp of different families, the number of families is not less than 10;

[0055] 2) Taking the paternal, maternal and sibling individuals of the juvenile shrimp in step 1) as target individuals, the genotype information of the target individuals is identified by using the steps in the application of the kit in genotype information identification;

[0056] S2 Mixed breeding

[0057] 3) In each family of step 1), 30-50 juvenile shrimps are randomly taken and evenly put into multiple parallel breeding ponds for mixed breeding, the water temperature is not less than 23℃, and the time is 60-90 days to obtain test individuals;

[0058] S3 Trait evaluation

[0059] 4) The number of test individuals in each breeding pond is counted, and the test individuals are weighed;

[0060] 5) The genotype information of the test individuals obtained in step 4) is identified by using the steps in the application of the kit in genotype information identification;

[0061] 6) The pedigree of the test individuals is reconstructed by using the genotype information of the test individuals obtained in step 5) and the genotype information of the target individuals obtained in step 2) to obtain the family information of the test individuals;

[0062] 7) According to the family information obtained in step 6), the number of survival and the weight of the test individuals in different families are counted, so as to obtain the survival traits and growth traits of different Penaeus vannamei family mixed culture.

[0063] Preferably, in the step 1), the larvae are in P15-P40 stage.

[0064] Preferably, in the step 2), the sibling individuals are in P45 stage or above.

[0065] Preferably, in the step 2), in the process of identifying the genotype information of the target individual: if the parents of the family are successfully genotyped, 1-3 sibling individuals are selected from the family for genotyping, so as to obtain the genotype information of the target individual; if the parents of the family have 1 individual that is not successfully genotyped, 2-4 sibling individuals are selected from the family for genotyping, so as to obtain the genotype information of the target individual; if the parents of the family are not successfully genotyped, 3-5 sibling individuals are selected from the family for genotyping, so as to obtain the genotype information of the target individual.

[0066] Preferably, in the step 3), the culture density of the larvae of each family in each culture tank is 100-300 tails / m 3 .

[0067] Preferably, in the step 6), in the case that the genotype information of the father and the mother in the target individual exists, the pedigree is generated by using the genotype information of the father and the mother in the test individual and the target individual, so as to obtain the family information to which the test individual belongs; in the case that the genotype information of the father and the mother in the target individual does not exist, the clustering analysis is carried out by using the genotype information of the sibling individuals in the test individual and the target individual, and the family information to which the test individual belongs is obtained according to the known family information of the sibling individuals in the target individual. Embodiment of the present application

[0068] Example one

[0069] The kit for evaluating the mixed culture traits of Penaeus vannamei family in the present application, the application of the kit in the identification of genotype information comprises the following steps:

[0070] 1) Extraction and detection of Penaeus vannamei genomic DNA

[0071] The muscle, swimming foot or antennae tissue of Penaeus vannamei is selected, and high-throughput DNA kit is used to extract DNA; the DNA sample after extraction is detected in two ways, one is to use 1% agarose gel electrophoresis method to analyze the purity and integrity of the DNA, and the other is to use Qubit fluorescence quantification instrument to accurately quantify the concentration of the DNA.

[0072] 2) Construction of DNA library

[0073] Taking the qualified DNA sample of quantitative quality inspection, using ultrasonic crusher for random physical crushing, the peak of the crushed fragment is controlled at 200-300bp, end repair, and A tail connection;

[0074] Using ligase to connect the A-added DNA fragments with sequencing adapters, using carboxyl-modified magnetic beads for purification and fragment selection, and retaining the 200-300bp insertion fragments in the connection product;

[0075] Adding sequencing primers with Barcode (molecular barcodes) and high-fidelity PCR (Polymerase Chain Reaction, PCR) reaction system to the connection product, PCR amplification, different Barcodes are used to distinguish different DNA samples, and carboxyl magnetic beads are used for purification to obtain purified library DNA.

[0076] 3) Construction of targeted sequencing library

[0077] Taking 500ng of purified library DNA, freeze-drying, adding probe mixture and hybrid capture reagent in the kit applied in the family mixed culture trait evaluation of Litopenaeus vannamei of the application, denaturation, incubation at 65℃ for 2h, and completing the hybrid capture reaction;

[0078] The hybrid capture product is washed with washing solution to remove DNA fragments of non-target regions and obtain target DNA fragments;

[0079] The obtained target DNA fragments are amplified and purified to construct a targeted sequencing library.

[0080] 4) DNA sequencing

[0081] After the construction of the targeted sequencing library, the Qubit 2.0 fluorescence quantifier is used for preliminary quantification, and the qPCR (Quantitative Real-time Polymerase Chain Reaction, real-time fluorescent quantitative PCR, referred to as real-time fluorescent quantitative PCR) method is used for accurate quantification of the effective concentration of the targeted sequencing library, so that the obtained targeted sequencing library is detected to be qualified, so as to ensure the quality of the library;

[0082] After the targeted sequencing library is detected to be qualified, DNA sequencing is performed by using the second-generation sequencing technology.

[0083] 5) Genotype analysis

[0084] Data quality control (removing adapters and low-quality data), aligning with the reference genome sequence of Litopenaeus vannamei, variation detection, and thus obtaining the genotype information of the tested Litopenaeus vannamei.

[0085] Example Two

[0086] Referring to FIG. 1, a kit for evaluating the traits of mixed breeding of Litopenaeus vannamei families, and the application of the kit in evaluating the traits of mixed breeding of Litopenaeus vannamei families, comprise the following steps:

[0087] S1 Preparation

[0088] 1) In July 2023, a G6 generation (breeding to the sixth generation) core breeding population of Litopenaeus vannamei was constructed, and 22 families were selected therefrom;

[0089] 2) The paternal, maternal and sibling individuals of the juvenile shrimp family were taken, 5 individuals with known family information were taken from each family as target individuals for subsequent family identification, the genotype information of the target individuals was identified using the method described in Example 1, and the genotype information of 74 sibling individuals and 15 target individuals of the maternal individuals in the 22 families was successfully detected;

[0090] S2 Mixed breeding

[0091] 3) In each family of step 1), 30 juvenile shrimp individuals with a growth stage of P15 were randomly taken, and were evenly placed into 3 parallel breeding ponds, with a breeding density of 110 individuals / m 3 , mixed breeding, water temperature was 28℃, time was 90 days, and test individuals were obtained;

[0092] S3 Trait evaluation

[0093] 4) The number of test individuals in each breeding pond was counted, and the test individuals were taken to measure the body weight;

[0094] 5) The genotype information of the test individuals obtained in step 4) was identified using the method described in Example 1;

[0095] 6) The 1K SNP genotype information of 292 individuals was finally obtained, and the genotype information was reconstructed and clustered with the genotype information of the target individuals obtained in step 2) by using the sequoia software package in R language;

[0096] 7) According to the family information obtained in step 6), the survival number and body weight of the test individuals in different families were counted, so as to obtain the survival traits and growth traits of different Litopenaeus vannamei family mixed breeding.

[0097] As can be seen from FIG. 2, the present application clearly distinguishes the survival rate between families. As can be seen from FIG. 3, the present application clearly distinguishes the harvest body weight between families. Industrial applicability

[0098] Therefore, compared with the prior art, the beneficial effects of the present application are: the present application uses SNP site combination for parentage identification, different families do not need to be bred separately, replaces the traditional VIE marker, realizes the mixed breeding of individuals among different families in early stage, effectively reduces the common environment effect caused by single family breeding in early stage, and improves the accuracy of Litopenaeus vannamei family mixed breeding trait evaluation; and compared with the traditional VIE marker for identifying family, the SNP site combination of the present application can accurately calculate the kinship between individuals, can accurately distinguish the family of Litopenaeus vannamei, and will not appear the phenomena of individual marker falling off, misplacement and human identification error, so as to obtain accurate pedigree. The evaluation method of the present application has low cost, and realizes the effective evaluation of the survival trait and growth trait of the Litopenaeus vannamei family mixed breeding.

[0099] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An application of a combination of SNPs in the evaluation of family-mixed culture traits of Litopenaeus vannamei, characterized in that: The SNP site combination includes 1125 SNP sites. The physical positions of the SNP sites are determined based on the comparison of the reference genome sequence of Penaeus vannamei. The information of the SNP sites is shown in Table 1, wherein ID is the site number, Scaffold is the scaffold name of the Penaeus vannamei reference genome where the site is located, Position is the physical position of the site on the scaffold, and Ref is the base name of the site in the Penaeus vannamei reference genome; Table 1 SNP locus information 2. A molecular probe for use in evaluating traits of mixed culture families of Litopenaeus vannamei, characterized in that: The molecular probe is a DNA single-stranded probe, and the molecular probe is the SNP described in the application of the SNP site combination of the vannamei shrimp in the evaluation of its family mixed breeding traits according to claim 1. The site is designed with 1-4 different nucleotide sequences, each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP site.

3. A kit for use in evaluating traits of mixed culture families of Litopenaeus vannamei, characterized in that: The kit comprises a probe mixture and a hybrid capture reagent, wherein the probe mixture is prepared from the molecular probe used in the evaluation of the mixed culture traits of the vannamei shrimp family according to claim 2.

4. Application of a kit in genotype information identification, characterized in that: The following steps are involved: 1) DNA of the tested Litopenaeus vannamei is sheared, end-repaired, adapter-ligated, and purified to obtain a sequencing library, and then the library is amplified and purified to obtain purified library DNA; 2) adding the probe mixture and hybridization capture reagent of the kit for use in the evaluation of traits of mixed culture of Litopenaeus vannamei families according to claim 3 to the purified library DNA obtained in step 1), performing hybridization capture, eluting, removing DNA fragments in non-target regions, obtaining target DNA fragments including the SNP site, amplifying and purifying the obtained target DNA, and constructing a targeted sequencing library; 3) The targeted sequencing library obtained in step 2) is tested. After passing the test, DNA sequencing is performed using second-generation sequencing technology, and the sequencing results are compared with the reference genome sequence of Penaeus vannamei to obtain the genotype information of the tested Penaeus vannamei.

5. Use of a kit for evaluating the traits of mixed culture of Litopenaeus vannamei families, characterized in that: The following steps are involved: S1 Preparation 1) Select shrimp larvae from different families, with the number of families not less than 10; 2) taking the paternal, maternal, and sibling individuals of the shrimp larvae in step 1) as target individuals, and identifying the genotype information of the target individuals using the steps of using the kit according to claim 4 for genotype information identification; S2 mixed farming 3) Randomly select 30-50 shrimp from each family in step 1) and place them in multiple parallel culture In the breeding pond, mixed culture is carried out, the water temperature is not lower than 23℃, and the time is 60-90 days, and the individual can be tested; S3 trait evaluation 4) Count the number of test individuals in each breeding pond, take out the test individuals, and measure their body weight; 5) using the test individual obtained in step 4) to identify the genotype information of the test individual using the steps of using the kit according to claim 4 for identifying genotype information; 6) Reconstructing a pedigree using the genotype information of the test individual obtained in step 5) and the genotype information of the target individual obtained in step 2) to obtain the family information to which the test individual belongs; 7) Based on the family information obtained in step 6), the number of survival and weight of the test individuals in different families are counted, thereby obtaining the survival and growth traits of mixed culture of different Vannamei shrimp families.

6. Use of the kit according to claim 5 in the evaluation of traits of mixed culture of Litopenaeus vannamei families, characterized in that: In the step 1), the shrimp larvae are at the P15-P40 stage.

7. Use of the kit according to claim 5 in the evaluation of traits of mixed culture of Litopenaeus vannamei families, characterized in that: In step 2), the sibling individuals are at the P45 stage or above.

8. Use of the kit according to claim 5 in the evaluation of traits of mixed culture of Litopenaeus vannamei families, characterized in that: In step 2), during the process of identifying the genotype information of the target individual: if the typing of the parents of the family is successful, 1-3 sibling individuals are selected from the family for genotyping, thereby obtaining the genotype information of the target individual; if one individual of the parents of the family is not successfully typed, 2-4 sibling individuals are selected from the family for genotyping, thereby obtaining the genotype information of the target individual; if typing of both parents of the family is not successful, 3-5 sibling individuals are selected from the family for genotyping, thereby obtaining the genotype information of the target individual.

9. Use of the kit according to claim 5 in the evaluation of traits of mixed culture of Litopenaeus vannamei families, characterized in that: In step 3), the stocking density of each family of shrimp in each breeding pond is 100-300 shrimp / m 3 .

10. Use of the kit according to claim 5 in the evaluation of traits of mixed culture of Litopenaeus vannamei families, characterized in that: In step 6), if the genotype information of the father and mother of the target individual exists, the genotype information of the father and mother of the test individual and the target individual is used to generate a pedigree to obtain the family information to which the test individual belongs; In the absence of genotype information of the father and mother of the target individual, cluster analysis is performed using the genotype information of the test individual and the siblings of the target individual, and the family information to which the test individual belongs is obtained based on the known family information of the siblings of the target individual.

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