A SNP marker combination and molecular probe, kit and application for sex identification of Litopenaeus vannamei

Abstract

The present invention proposes a SNP marker combination and a molecular probe, a kit and an application for sex identification of Penaeus vannamei, belonging to the technical field of SNP molecular markers. The SNP marker combination of the present invention includes 10 SNP markers, and the physical positions of the SNP markers are determined based on the comparison of the reference genome sequence of Penaeus vannamei. The present invention also provides a molecular probe for capturing the above-mentioned SNP marker combination, a kit including the molecular probe, and the application of the kit in the determination of genotype information, establishment of a sex prediction model and sex identification of Penaeus vannamei. The number of SNP markers of the present invention is appropriate, the distance between SNP markers is appropriate, and they are distributed on multiple Scaffolds, which are widely representative. The genotype information of these SNP markers has a good correlation with sex. The kit developed by the present invention is simple to operate when performing sex identification of Penaeus vannamei, has a high accuracy rate, and has reliable results.

Description

Technical Field

[0001] The invention relates to the technical field of SNP molecular markers, and in particular to a SNP marker combination and a molecular probe, a kit and an application thereof for sex identification of Litopenaeus vannamei. Background Art

[0002] Litopenaeus vannamei ( Litopenaeus vannamei ), also known as the South American white shrimp, also known as white-foot shrimp and Vannamei shrimp, is native to the waters of the Pacific coast of Central and South America. The growth climate zone is tropical, subtropical, warm temperate, and temperate waters. It is distributed from the west coast of the Pacific Ocean to the central Gulf of Mexico. It is one of the three most productive shrimp species in the world. In 2023, the aquaculture output of Vannamei shrimp in China exceeded 2.2 million tons, ranking first in the world. In the middle and late stages of the breeding process of Vannamei shrimp, the growth rate of female individuals is significantly higher than that of male individuals. Therefore, it is of great significance to explore the mechanism of sex regulation of Vannamei shrimp and to achieve single-sex control of Vannamei shrimp.

[0003] The traditional method for sex identification of Penaeus vannamei is manual identification, which is time-consuming and labor-intensive. Moreover, for young shrimp weighing less than 5g, the error rate of manual identification is very high. In the early stages of the development of Penaeus vannamei, the sex of individuals can be accurately identified by molecular markers, and targeted tracking experiments on their sex development can be conducted to regulate the male-female ratio of Penaeus vannamei during the breeding process, thereby improving breeding efficiency. On March 17, 2020, Chinese patent CN110885882A disclosed a method for simultaneously identifying the genetic sex of Penaeus vannamei at the DNA or RNA level. By using three SNP markers that differ between males and females on sex-related genes and typing these SNPs, sex identification can be achieved through the DNA or RNA of individuals. This method for identifying the sex of Penaeus vannamei only uses three SNP markers. The number of markers is too small, and the maximum distance between markers is only 304bp. The distance between markers is short, the dependence between markers is strong, and the representativeness is poor. When determining the genotype information, once the base at the genome position where the marker is located mutates, the genotype information will be incorrect. At the same time, if the fragment of the genome to be tested is missing, the genotype information of the three SNP markers cannot be obtained, making it difficult to accurately identify the sex of Penaeus vannamei. Especially when the sex of a large number of Penaeus vannamei individuals is identified, the probability of errors in the identification results is even greater. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP marker combination and a molecular probe, a kit and an application for sex identification of Penaeus vannamei, aiming to solve the problem that the sex identification method of Penaeus vannamei in the prior art is too small in number and the distance between the markers is short, so that the mutual dependence is strong and the markers are not representative. When the genotype information is determined, it is easy to have genotype information errors or the genotype information of these SNP markers cannot be obtained, thereby reducing the accuracy of the sex identification result.

[0005] In order to solve the above technical problems, the technical solution of the present invention is achieved as follows:

[0006] The present invention discloses a SNP marker combination for sex identification of Penaeus vannamei. The SNP marker combination includes 10 SNP markers. The physical positions of the SNP markers are determined based on the comparison of the reference genome sequence of Penaeus vannamei. The information of the SNP markers is shown in Table 1, wherein ID is the SNP marker number, Scaffold is the name of the Scaffold of the reference genome of Penaeus vannamei where the SNP marker is located, Position is the physical position of the SNP marker on the Scaffold, Ref is the name of the base of the reference genome of Penaeus vannamei marked by the SNP, and Alt is the name of the mutant base of the reference genome of Penaeus vannamei marked by the SNP.

[0007] Table 1 Information of SNP marker combinations

[0008]

[0009] The SNP marker combination for sex identification of Penaeus vannamei of the present invention is composed of 10 SNP markers. The SNP marker combination is based on GWAS (Genome-wide association studies, i.e., whole genome association studies) screened 10 SNP markers with the highest sex correlation. The number of the SNP marker combination is appropriate, the distance between the SNP markers is appropriate, and the SNP markers are distributed on multiple scaffolds, which is widely representative. The genotype information of the SNP markers is well correlated with the sex, and the result of sex identification is more robust. The accuracy of sex identification of Penaeus vannamei is greatly improved, and the method is particularly suitable for sex identification of a large number of Penaeus vannamei individuals. The problem of low sex identification accuracy in a Penaeus vannamei sex identification method using 3 SNP markers in the prior art is solved, and the sex identification needs of different Penaeus vannamei populations are met. The method has wide applicability and reliability in sex identification of diversified Penaeus vannamei populations.

[0010] SNP, Single Nucleotide Polymorphism, mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. DNA, DeoxyriboNucleic Acid, is one of the four biological macromolecules contained in biological cells, one of the nucleic acids. RNA, Ribonucleic Acid, is a genetic information carrier present in biological cells and some viruses and viroids. ID, identification, tag number; Scaffold, scaffold; Position, physical position; Ref, reference, reference genome base name; Alt, alternative, mutant base name; The website for obtaining the reference genome sequence of Penaeus vannamei in the present invention is: https: / / www.ncbi.nlm.nih.gov / data-hub / genome / GCF_003789085.1 / , where the version number of the reference genome sequence of Penaeus vannamei is GCF_003789085.1.

[0011] The present invention discloses a molecular probe for sex identification of Penaeus vannamei. The molecular probe is a single-stranded DNA probe. The molecular probe is 1 to 4 different nucleotide sequences designed based on the SNP markers in the SNP marker combination for sex identification of Penaeus vannamei described above. Each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP marker.

[0012] The molecular probe of the present invention is a molecular probe for detecting the combination of SNP markers of Penaeus vannamei. This molecular probe combination can perform fixed-point capture on 10 target areas. Single-stranded nucleotides are synthesized according to the nucleotide sequence designed above. The synthesized length is 110 bp, and the DNA nucleotide sequence modified with a biotin group at the 5' end is used as the molecular probe; the synthesized molecular probes are mixed with equal molar mass, and a probe mixture is prepared using a mixed solution of EDTA and Tris-HCL. 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; bp, base pair, is a pair of matching bases in DNA and RNA, connected by hydrogen bonds, and is the basic unit for measuring the length of DNA and RNA.

[0013] The invention discloses a kit for sex identification of Penaeus vannamei. The kit comprises a probe mixture and a hybrid capture reagent. The probe mixture is prepared according to the molecular probe for sex identification of Penaeus vannamei described above.

[0014] The kit for sex identification of Litopenaeus vannamei of the present invention consists of a probe mixture and a hybrid capture reagent. The probe mixture is prepared from a molecular probe. The hybrid capture reagent usually consists of Block I (blocker I), Block II (blocker II), 2× Hyb Buffer (2 times hybridization buffer), Hyb Buffer Enhancer (hybridization buffer enhancer), Primer Mix (primer mixture), PCR Master Mix (PCR master mix), 2× Beads Wash Buffer (2 times magnetic bead washing buffer), 10× Wash Buffer I (10 times washing buffer I), 10× Wash Buffer II (10 times washing buffer II), 10× Wash Buffer III (10 times washing buffer III), 10× Strigent Wash Buffer (10 times stringent washing buffer), DNA Probe Beads (DNA probe magnetic beads), and DNA Clean Beads (DNA purification magnetic beads). The hybrid capture reagents are preferably from Shijiazhuang Boridi Biotechnology Co., Ltd. The kit has low cost and good accuracy.

[0015] The invention discloses an application of a kit for sex identification of Penaeus vannamei in determining genotype information of Penaeus vannamei, comprising the following steps: 1) breaking, end-repairing, linker-connecting and purifying DNA of a tested Penaeus vannamei to obtain a sequencing library, and then amplifying and purifying the library to obtain purified library DNA; 2) adding a probe mixture and a hybridization capture reagent in the kit for sex identification of Penaeus vannamei described above to the purified library DNA obtained in step 1) to perform hybridization capture, eluting, removing DNA fragments in non-target regions, obtaining target DNA fragments including the SNP marker, amplifying and purifying the obtained target DNA, and constructing a targeted sequencing library; 3) testing the targeted sequencing library obtained in step 2), and after the testing is qualified, performing DNA sequencing using a second-generation sequencing technology, and comparing the sequencing results on a reference genome sequence of Penaeus vannamei, thereby obtaining genotype information of the tested Penaeus vannamei.

[0016] The kit for sex identification of Penaeus vannamei of the present invention can be used to determine the genotype information of Penaeus vannamei, and is a method for genotyping the Penaeus vannamei genome based on targeted capture sequencing, which uses the molecular probe of the present invention to capture the target region segment in a targeted manner, and the target region segment refers to a DNA fragment including a target marker, thereby obtaining the target DNA fragment, and finally obtaining the genotype information of the tested Penaeus vannamei after DNA sequencing and comparison with the reference genome sequence of Penaeus vannamei. This genotyping method is not only simple to operate, but also can maintain the consistency of site selection in sequencing of different batches; at the same time, it also has the advantages of low cost and flexible site selection.

[0017] The invention discloses an application of a kit for sex identification of Penaeus vannamei in establishing a sex prediction model of Penaeus vannamei, comprising the following steps: a) selecting Penaeus vannamei, constructing a training group, manually identifying the sex of the Penaeus vannamei, and performing digital coding, wherein the male is marked as 1 and the female is marked as 0; b) using the steps of the application of the kit for sex identification of Penaeus vannamei in determining genotype information of Penaeus vannamei in the above-mentioned method to determine the genotype information of Penaeus vannamei, and performing digital coding, wherein, compared with a reference genome of Penaeus vannamei, a homozygous genotype with the same base is marked as 0, a heterozygous genotype is marked as 1, and a homozygous genotype with a variant base is marked as 2; c) using the sex information of the Penaeus vannamei training group after digital coding in step a) as a dependent variable, and using the genotype information of the Penaeus vannamei training group after digital coding in step b) as an independent variable, to establish a random forest classification prediction model for sex identification of Penaeus vannamei.

[0018] The kit for sex identification of Penaeus vannamei of the present invention can be used to establish a sex prediction model for Penaeus vannamei, and this prediction model is used to identify the sex information of Penaeus vannamei. In the sex identification process of Penaeus vannamei, the genotype information of Penaeus vannamei is determined by using the SNP marker combination of the present invention, and a random forest classification prediction model between the sex information of Penaeus vannamei and its genotype information is constructed. This random forest classification prediction model has wide versatility, and the sex information of Penaeus vannamei to be tested can be successfully obtained through the random forest classification prediction model. The establishment of the random forest classification prediction model in the present invention further improves the effectiveness of the genotype information of the above-mentioned SNP markers in the sex identification process of Penaeus vannamei, thereby further improving the accuracy of the sex identification results of Penaeus vannamei, and is particularly suitable for sex identification of a large number of Penaeus vannamei individuals.

[0019] As a preferred implementation scheme, in the step c), when the random forest classification prediction model is established, the training population is the training set, and the Bootstrap method is used for sampling to obtain a training subset. According to the independent variables and dependent variables of the training subset, a single decision tree is constructed, and a total of 100-200 samplings are performed to generate a decision tree group and establish a random forest classification prediction model. The Bootstrap method, also known as the Bootstrap Method, is a uniform sampling method with replacement from a given training set. When the random forest classification prediction model of the present invention is established, the sample extraction amount is appropriate and the representativeness is strong, and the obtained random forest classification prediction model has a wide range of applicability.

[0020] As a preferred embodiment, in the step a), the number of Penaeus vannamei is not less than 1000. In the process of establishing the random forest classification prediction model, a considerable number of Penaeus vannamei is selected as the training population. This number of Penaeus vannamei is widely representative, and the random forest classification prediction model obtained therefrom has better applicability.

[0021] As a preferred embodiment, in the step a), the weight of the vannamei shrimp is not less than 7 g / tail. In the process of establishing the random forest classification prediction model of the present invention, in order to improve the accuracy of the results of manual sex identification of vannamei shrimp, vannamei shrimp with a weight of more than 7 g / tail are usually selected as the training group; such vannamei shrimp are of suitable size, and the results of manual sex identification are accurate, which further improves the accuracy of the random forest classification prediction model established by the shrimp.

[0022] The invention discloses an application of a kit for sex identification of Penaeus vannamei in sex identification of Penaeus vannamei, comprising the following steps: S1, taking Penaeus vannamei to be tested, determining genotype information of the Penaeus vannamei to be tested by using the steps of the application of the kit for sex identification of Penaeus vannamei in genotype information determination of Penaeus vannamei described above, and performing digital coding, wherein, compared with a reference genome of Penaeus vannamei, homozygous genotypes with identical bases are recorded as 0, heterozygous genotypes are recorded as 1, and homozygous genotypes with variant bases are recorded as 2; S2, using the digitally coded genotype information of the Penaeus vannamei to be tested obtained in step S1 as an independent variable, inputting it into a random forest classification prediction model obtained by applying any one of the above-mentioned kits for sex identification of Penaeus vannamei in establishing a sex prediction model of Penaeus vannamei, if the output is 1, it is male, if the output is 0, it is female, and the sex information of the Penaeus vannamei to be tested is obtained.

[0023] The kit of the present invention can be used to accurately identify the sex information of Penaeus vannamei. In the process of Penaeus vannamei sex identification, it is only necessary to use the kit of the present invention to determine the genotype information of the Penaeus vannamei to be tested, and then input it into the random forest classification prediction model that has been established by the present invention to successfully obtain the sex information of the Penaeus vannamei to be tested. The Penaeus vannamei sex identification method of the present invention combines the genotype information of 10 SNP markers closely related to sex identification with the random forest classification prediction model to ensure the accuracy of the Penaeus vannamei sex identification result, and is particularly suitable for the sex identification of a large number of Penaeus vannamei individuals. This Penaeus vannamei sex identification method is simple to operate, can be used in different Penaeus vannamei populations, has a wide range of applicability, an accuracy rate of 100%, and a reliable identification result.

[0024] As a preferred embodiment, in the step S1, the weight of the Penaeus vannamei shrimp to be tested is not less than 2 g / tail. The method for identifying the sex of Penaeus vannamei shrimp of the present invention is suitable for all Penaeus vannamei shrimp populations, especially Penaeus vannamei shrimp populations with a weight of more than 2 g / tail; and after the sex identification, the continued growth, breeding and sale of the Penaeus vannamei shrimp population will not be affected.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: the number of SNP marker combinations for sex identification of Penaeus vannamei of the present invention is appropriate, the distance between SNP markers is appropriate, and they are distributed on multiple Scaffolds, which are widely representative, so that the genotype information of these SNP markers has a good correlation with the sex. When the kit developed using this SNP marker combination is used for sex identification of Penaeus vannamei, a random forest classification prediction model is first established. It is only necessary to use the kit of the present invention to determine the genotype information of the Penaeus vannamei to be tested, and input it into the above-mentioned established random forest classification prediction model, so that the sex information of the Penaeus vannamei to be tested can be successfully obtained. The method for sex identification of Penaeus vannamei is simple to operate, can be used in different Penaeus vannamei populations, and has wide applicability. Under the combined effect of the above-mentioned SNP marker combination and the random forest classification prediction model, the sex identification result is more robust, and the accuracy of the sex identification result reaches 100%. It is particularly suitable for sex identification of a large number of Penaeus vannamei individuals, solves the problem of low sex identification accuracy in the Penaeus vannamei sex identification method using 3 SNP markers in the prior art, meets the sex identification needs of different Penaeus vannamei populations, and has wide applicability and reliability in sex identification of diversified Penaeus vannamei populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of the application of the kit provided by the present invention in the sex identification of Penaeus vannamei. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The present invention discloses a SNP marker combination for sex identification of Penaeus vannamei. The SNP marker combination includes 10 SNP markers. The physical positions of the SNP markers are determined based on the comparison of the reference genome sequence of Penaeus vannamei. The information of the SNP markers is shown in Table 1, wherein ID is the SNP marker number, Scaffold is the name of the Scaffold of the reference genome of Penaeus vannamei where the SNP marker is located, Position is the physical position of the SNP marker on the Scaffold, Ref is the name of the base of the reference genome of Penaeus vannamei marked by the SNP, and Alt is the name of the mutant base of the reference genome of Penaeus vannamei marked by the SNP.

[0029] The present invention discloses a molecular probe for sex identification of Penaeus vannamei. The molecular probe is a single-stranded DNA probe. The molecular probe is 1 to 4 different nucleotide sequences designed based on the SNP markers in the SNP marker combination for sex identification of Penaeus vannamei described above. Each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP marker.

[0030] The invention discloses a kit for sex identification of Penaeus vannamei. The kit comprises a probe mixture and a hybrid capture reagent. The probe mixture is prepared according to the molecular probe for sex identification of Penaeus vannamei described above.

[0031] The application of a kit for sex identification of Penaeus vannamei of the present invention in determining genotype information of Penaeus vannamei comprises the following steps:

[0032] 1) The DNA of the test shrimp Penaeus 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;

[0033] 2) adding the probe mixture and hybrid capture reagent in the kit for sex identification of Litopenaeus vannamei described above to the purified library DNA obtained in step 1), performing hybrid capture, eluting, removing DNA fragments in non-target regions, obtaining target DNA fragments including the SNP marker, amplifying and purifying the obtained target DNA, and constructing a targeted sequencing library;

[0034] 3) The targeted sequencing library obtained in step 2) is tested. If the test is qualified, DNA sequencing is performed using the 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.

[0035] The application of a kit for sex identification of Penaeus vannamei of the present invention in establishing a sex prediction model of Penaeus vannamei comprises the following steps:

[0036] a) Selecting Penaeus vannamei, constructing a training population, manually identifying their sex, and performing digital coding, wherein males are marked as 1 and females are marked as 0;

[0037] b) using the steps of the application of the kit for sex identification of Penaeus vannamei in the determination of genotype information of Penaeus vannamei as described above to determine the genotype information of Penaeus vannamei, and performing digital coding, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with variant base is recorded as 2;

[0038] c) Using the sex information of the Penaeus vannamei training population after digital coding in step a) as the dependent variable and the genotype information of the Penaeus vannamei training population after digital coding in step b) as the independent variable, a random forest classification prediction model for sex identification of Penaeus vannamei is established.

[0039] Preferably, in the step c), when the random forest classification prediction model is established, the training population is the training set, and the Bootstrap method is used for sampling to obtain a training subset. A single decision tree is constructed based on the independent variables and dependent variables of the training subset. The sampling is performed 100-200 times in total to generate a decision tree group and establish a random forest classification prediction model.

[0040] Preferably, in step a), the number of Penaeus vannamei shrimps is not less than 1000.

[0041] Preferably, in step a), the weight of each Litopenaeus vannamei shrimp is not less than 7 g.

[0042] The application of a kit for sex identification of Penaeus vannamei of the present invention in sex identification of Penaeus vannamei comprises the following steps:

[0043] S1: taking a Penaeus vannamei shrimp to be tested, determining the genotype information of the Penaeus vannamei shrimp to be tested using the steps of the application of the kit for sex identification of Penaeus vannamei in the determination of genotype information of Penaeus vannamei as described above, and performing digital coding, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with a variant base is recorded as 2;

[0044] S2 uses the digitally encoded genotype information of the Penaeus vannamei to be tested obtained in step S1 as an independent variable, and inputs it into the random forest classification prediction model obtained by applying the kit for sex identification of Penaeus vannamei described in any one of the above items in the establishment of a sex prediction model for Penaeus vannamei. If the output is 1, it is male, and if the output is 0, it is female, the sex information of the Penaeus vannamei to be tested is obtained.

[0045] Preferably, in step S1, the weight of each of the Penaeus vannamei shrimp to be tested is not less than 2 g.

[0046] Embodiment 1

[0047] A method for obtaining a SNP marker combination for sex identification of Litopenaeus vannamei of the present invention comprises the following steps:

[0048] 1) Sample collection and genotyping

[0049] A total of 1244 Penaeus vannamei shrimp were selected, including 621 males and 623 females. Muscle tissues were collected and stored in 5 mL sampling tubes containing DNA preservation solution at room temperature.

[0050] High-purity DNA was extracted using a DNA extraction kit, and a targeted genotype detection technology based on liquid phase probe hybridization was used to construct a liquid phase chip "Huanghaixin No. 1" 55K SNP-marked targeted sequencing library;

[0051] The sequencing and bioinformatics analysis of the DNBSEQ-T7 gene sequencer (formerly known as the MGISEQ-T7 sequencing platform) produced by MGI revealed 56,214 SNP loci in 1,244 Litopenaeus vannamei.

[0052] 2) Determination of sex-related SNP marker combination

[0053] The SNP sites obtained in step 1) were subjected to quality control, including filtering of the minor allele frequency and site typing success rate, ensuring that the minor allele frequency was greater than 0.01 and the SNP site detection rate was greater than 0.9. After quality control, a total of 50,174 high-quality SNP sites were obtained;

[0054] According to the sex information of each individual Penaeus vannamei, rMVP software was used for analysis, and a mixed linear model was used to identify SNP loci closely related to sex. The 10 SNP loci with the highest sex correlation were selected and listed in Table 1, thus obtaining the SNP marker combination for sex identification of Penaeus vannamei.

[0055] Embodiment 2

[0056] The method for preparing a kit for sex identification of Litopenaeus vannamei of the present invention comprises the following steps:

[0057] 1) Preparation of probe mixture

[0058] According to the SNP markers in the SNP marker combination for sex identification of Litopenaeus vannamei obtained in Example 1, 4 different nucleotide sequences were designed, each nucleotide sequence was 110 bp, and the average coverage range was 55 bp upstream and downstream of the selected SNP marker;

[0059] Single-stranded nucleotides were synthesized according to the nucleotide sequence designed above, and the synthesized length was 110 bp. The DNA nucleotide sequence modified with a biotin group at the 5' end was used as a molecular probe;

[0060] The synthesized molecular probes are mixed in equal molar mass, and a probe mixture is prepared by using a mixture of EDTA and Tris-HCL, and then packaged.

[0061] 2) Preparation of hybrid capture reagent

[0062] Block I (blocker I), Block II (blocker II), 2× Hyb Buffer (2x hybridization buffer), Hyb Buffer Enhancer (hybridization buffer enhancer), Primer Mix (primer mixture), PCR Master Mix (PCR master mix), 2× Beads Wash Buffer (2x magnetic beads washing buffer), 10× Wash Buffer I (10x washing buffer I), 10× Wash Buffer II (10x washing buffer II), 10× Wash Buffer III (10x washing buffer III), 10× Strigent Wash Buffer (10x stringent washing buffer), DNA Probe Beads (DNA probe magnetic beads), and DNA Clean Beads (DNA purification magnetic beads) are prepared to form a hybridization capture reagent and packaged.

[0063] 3) Preparation of the kit

[0064] The probe mixture obtained in step 1) and the hybridization capture reagent obtained in step 2) are placed in the same outer packaging box to obtain a kit for sex identification of Penaeus vannamei.

[0065] Embodiment 3

[0066] The application of a kit for sex identification of Penaeus vannamei of the present invention in determining genotype information of Penaeus vannamei comprises the following steps:

[0067] 1) DNA extraction and detection

[0068] The tentacles of Penaeus vannamei were selected as the sampling tissue, and DNA was extracted using a high-throughput DNA kit; the extracted DNA samples were subjected to two tests, one was to analyze the purity and integrity of the DNA using 1% agarose gel electrophoresis, and the other was to accurately quantify the concentration of the DNA using a Qubit fluorescence quantifier.

[0069] 2) Construction of DNA library

[0070] Take the qualified DNA samples of quantitative quality inspection, use ultrasonic crusher to randomly break them physically, control the peak value of broken fragments to 200-300bp, repair the ends, and connect A tails;

[0071] The DNA fragments with A added were connected to the sequencing adapters using ligase, and the carboxyl-modified magnetic beads were used for purification and fragment selection, retaining the connection products with insert fragments of 200-300 bp;

[0072] Sequencing primers with barcodes (molecular barcodes) and a high-fidelity PCR (Polymerase Chain Reaction) reaction system are added to the ligation product for PCR amplification. Different barcodes are used to distinguish different DNA samples. Carboxyl magnetic beads are used for purification to obtain purified library DNA.

[0073] 3) Construction of targeted sequencing library

[0074] 500 ng of the purified library DNA was taken, freeze-dried, added to the probe mixture and hybrid capture reagent in the kit for sex identification of Litopenaeus vannamei described in Example 2, denatured, and incubated at 65° C. for 2 h to complete the hybrid capture reaction;

[0075] The hybrid capture product is washed with a washing solution to remove DNA fragments in non-target areas and obtain target DNA fragments;

[0076] The obtained target DNA fragments are amplified and purified to construct a targeted sequencing library.

[0077] 4) DNA sequencing

[0078] After the construction of the targeted sequencing library is completed, the Qubit 2.0 fluorescence quantifier is used for preliminary quantification, and then the qPCR (Quantitative Real-time Polymerase Chain Reaction, real-time fluorescence quantitative polymerase chain reaction, referred to as real-time fluorescence quantitative PCR) method is used to accurately quantify the effective concentration of the targeted sequencing library, so that the obtained targeted sequencing library is qualified for detection to ensure the quality of the library;

[0079] After the targeted sequencing library is qualified, DNA sequencing is performed using the second-generation sequencing technology.

[0080] 5) Genotype information analysis

[0081] After data quality control (removal of adapters and low-quality data), the sequencing results were aligned with the reference genome sequence of Penaeus vannamei to obtain the genotype information of the tested Penaeus vannamei.

[0082] Embodiment 4

[0083] The application of a kit for sex identification of Penaeus vannamei of the present invention in establishing a sex prediction model of Penaeus vannamei comprises the following steps:

[0084] a) 1244 shrimps of the Weifang strain of Shandong Province with a body weight of more than 7 g were selected to construct a training population. The shrimps were identified by hand, with 621 males and 623 females, and digitally coded, with males marked as 1 and females as 0;

[0085] b) using the steps of the application of the kit for sex identification of Penaeus vannamei in the determination of genotype information of Penaeus vannamei described in Example 3 to determine the genotype information of Penaeus vannamei training population, and performing digital coding, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with variant base is recorded as 2;

[0086] c) using the sex information of the training population of Penaeus vannamei after digital coding in step a) as the dependent variable, using the genotype information of the training population of Penaeus vannamei after digital coding in step b) as the independent variable, the training population as the training set, sampling by the Bootstrap method to obtain a training subset, and constructing a single decision tree according to the independent variable and dependent variable of the training subset;

[0087] Each time a node splits, the marker with the smallest Gini index and the corresponding genotype value threshold are selected from the three randomly selected SNP markers as the basis for splitting. The node splitting process continues until all training samples of a node belong to the same category or cannot be further split. At this time, a leaf node is generated, and finally a complete decision tree is constructed.

[0088] A total of 200 samplings were performed to generate a decision tree group consisting of 200 independent decision trees. Based on the decision tree group consisting of 200 independent decision trees, the association between different genotype information (0, 1, 2) and its different sex information (0, 1) was constructed at a specific SNP site to form a random forest classification prediction model for sex identification of Penaeus vannamei.

[0089] In the process of establishing the random forest classification prediction model for sex identification of Penaeus vannamei, the distribution of the association weights of different SNP markers and sex information is shown in Table 2, where ID is the marker number, Scaffold is the name of the Scaffold of the reference genome of Penaeus vannamei where the marker is located, Position is the physical position of the marker on the Scaffold, and Weight is the association weight of the SNP marker and sex;

[0090] Table 2 Distribution of weights associated with different SNP markers and gender

[0091]

[0092] Changes in the genotype information of SNP markers may affect the gender information of individuals. It can be seen from Table 2 that the weights of different SNP markers associated with gender are different. The weight value of each SNP site represents the strength of its association with gender. Among them, the weight values ​​of markers numbered A5, A7, A8, A9 and A10 are relatively large, all close to or much greater than 10%, which indicates that the association strength between these markers and gender is relatively large and they are the key factors in gender determination; the weight values ​​of markers numbered A1, A2, A3, A4 and A6 are relatively small, all less than 5%, which indicates that the association strength between these markers and gender is relatively small.

[0093] Embodiment 5

[0094] The application of a kit for sex identification of Penaeus vannamei of the present invention in sex identification of Penaeus vannamei comprises the following steps:

[0095] S1: 196 Penaeus vannamei of strain A in Wenzhou City, Zhejiang Province, including 96 females and 100 males, are taken as Penaeus vannamei to be tested, and the genotype information of the Penaeus vannamei to be tested is determined by using the steps of the application of the kit for sex identification of Penaeus vannamei in the determination of genotype information of Penaeus vannamei described in Example 3, and digital encoding is performed, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with variant base is recorded as 2;

[0096] S2 uses the digitally encoded genotype information of the tested Penaeus vannamei obtained in step S1 as an independent variable and inputs it into the random forest classification prediction model obtained in Example 4. If the output is 1, it is male, and if the output is 0, it is female, the gender information of the tested Penaeus vannamei is obtained.

[0097] Embodiment 6

[0098] On the basis of Example 5, Penaeus vannamei of strain B from Dongfang City, Hainan Province was selected as the Penaeus vannamei to be tested. A total of 124 Penaeus vannamei of this strain were selected, including 63 females and 61 males.

[0099] Embodiment 7

[0100] On the basis of Example 5, Penaeus vannamei of strain C from Qingdao City, Shandong Province was selected as the Penaeus vannamei to be tested. There were 63 Penaeus vannamei of this strain, all of which were female.

[0101] Embodiment 8

[0102] On the basis of Example 5, the vannamei shrimp of the D strain from Dongfang City, Hainan Province was selected as the vannamei shrimp to be tested. There were 61 vannamei shrimps of this strain, including 31 females and 30 males.

[0103] Table 3 Statistics of sex identification results of different populations of Penaeus vannamei

[0104]

[0105] The sex identification results obtained in Examples 5 to 8 are summarized in Table 3. As can be seen from Table 3, the identification results obtained by the sex identification method of Penaeus vannamei of the present invention are completely consistent with the sex information of Penaeus vannamei identified manually. Therefore, the accuracy of the results obtained by the sex identification method of Penaeus vannamei of the present invention is 100%. Moreover, the identification results obtained by the sex identification method of Penaeus vannamei of the present invention are not affected by the strain of the Penaeus vannamei population, nor by the number of the Penaeus vannamei population.

[0106] Therefore, compared with the prior art, the beneficial effects of the present invention are as follows: the number of SNP marker combinations for sex identification of Penaeus vannamei of the present invention is appropriate, the distance between SNP markers is appropriate, and they are distributed on multiple Scaffolds, which are widely representative, so that the genotype information of these SNP markers has a good correlation with the sex. When the kit developed using this SNP marker combination is used for sex identification of Penaeus vannamei, a random forest classification prediction model is first established. It is only necessary to use the kit of the present invention to determine the genotype information of the Penaeus vannamei to be tested, and input it into the above-mentioned established random forest classification prediction model to successfully obtain the sex information of the Penaeus vannamei to be tested. The method for sex identification of Penaeus vannamei is simple to operate, can be used in different Penaeus vannamei populations, and has wide applicability. Under the combined effect of the above-mentioned SNP marker combination and the random forest classification prediction model, the sex identification result is more robust, and the accuracy of the sex identification result reaches 100%. It is particularly suitable for sex identification of a large number of Penaeus vannamei individuals, solves the problem of low sex identification accuracy in the Penaeus vannamei sex identification method using 3 SNP markers in the prior art, meets the sex identification needs of different Penaeus vannamei populations, and has wide applicability and reliability in sex identification of diversified Penaeus vannamei populations.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of a SNP marker combination of Litopenaeus vannamei in sex identification thereof, characterized in that: The SNP marker combination includes 10 SNP markers, the physical positions of the SNP markers are determined based on the comparison of the reference genome sequence of Penaeus vannamei, and the information of the SNP markers is shown in Table 1, wherein ID is the SNP marker number, Scaffold is the name of the Scaffold of the reference genome of Penaeus vannamei where the SNP marker is located, Position is the physical position of the SNP marker on the Scaffold, Ref is the name of the base of the reference genome of Penaeus vannamei marked by the SNP, and Alt is the name of the mutant base of the reference genome of Penaeus vannamei marked by the SNP; Table 1 Information of SNP marker combinations 。 2. A molecular probe for sex identification of Penaeus vannamei, characterized in that: The molecular probe is a single-stranded DNA probe, which is 1-4 differential nucleotide sequences designed according to the SNP marker combination of Litopenaeus vannamei in its sex identification according to claim 1, each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP marker.

3. A kit for sex identification of Penaeus 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 for sex identification of Penaeus vannamei according to claim 2.

4. Use of a kit for sex identification of Penaeus vannamei in determining genotype information of Penaeus vannamei, characterized in that: The following steps are involved: 1) The DNA of the test shrimp Penaeus 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 hybrid capture reagent in the kit for sex identification of Litopenaeus vannamei according to claim 3 to the purified library DNA obtained in step 1), performing hybrid capture, eluting, removing DNA fragments in non-target regions, obtaining target DNA fragments including the SNP marker, amplifying and purifying the obtained target DNA, and constructing a targeted sequencing library; 3) The targeted sequencing library obtained in step 2) is tested. If the test is qualified, DNA sequencing is performed using the 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 sex identification of Penaeus vannamei in establishing a sex prediction model for Penaeus vannamei, characterized in that: The following steps are involved: a) Selecting Penaeus vannamei, constructing a training population, manually identifying their sex, and performing digital coding, wherein males are marked as 1 and females are marked as 0; b) using the step of using the kit for sex identification of Penaeus vannamei according to claim 4 in the determination of genotype information of Penaeus vannamei to determine the genotype information of Penaeus vannamei training population, and performing digital coding, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with variant base is recorded as 2; c) Using the sex information of the Penaeus vannamei training population after digital coding in step a) as the dependent variable and the genotype information of the Penaeus vannamei training population after digital coding in step b) as the independent variable, a random forest classification prediction model for sex identification of Penaeus vannamei is established.

6. The use of the kit for sex identification of Penaeus vannamei according to claim 5 in establishing a sex prediction model for Penaeus vannamei, characterized in that: In the step c), when the random forest classification prediction model is established, the training population is the training set, and the Bootstrap method is used for sampling to obtain the training subset. A single decision tree is constructed based on the independent variables and dependent variables of the training subset. The sampling is performed 100-200 times in total to generate a decision tree group and establish the random forest classification prediction model.

7. Use of the kit for sex identification of Penaeus vannamei according to claim 5 in establishing a sex prediction model for Penaeus vannamei, characterized in that: In the step a), the number of Penaeus vannamei shrimps is not less than 1000.

8. The use of the kit for sex identification of Penaeus vannamei according to claim 5 in establishing a sex prediction model for Penaeus vannamei, characterized in that: In the step a), the weight of each Litopenaeus vannamei shrimp is not less than 7 g.

9. Use of a kit for sex identification of Penaeus vannamei in sex identification of Penaeus vannamei, characterized in that: The following steps are involved: S1: taking a Penaeus vannamei shrimp to be tested, and determining the genotype information of the Penaeus vannamei shrimp to be tested using the step of applying the kit for sex identification of Penaeus vannamei in determining the genotype information of Penaeus vannamei according to claim 4, and performing digital coding, wherein, compared with the reference genome of Penaeus vannamei, the homozygous genotype with the same base is recorded as 0, the heterozygous genotype is recorded as 1, and the homozygous genotype with a variant base is recorded as 2; S2 uses the digitally encoded genotype information of the Penaeus vannamei to be tested obtained in step S1 as an independent variable, and inputs it into a random forest classification prediction model obtained by applying the kit for sex identification of Penaeus vannamei according to any one of claims 5 to 8 in establishing a sex prediction model for Penaeus vannamei. If the output is 1, it is male, and if the output is 0, it is female, the sex information of the Penaeus vannamei to be tested is obtained.

10. Use of the kit for sex identification of Penaeus vannamei according to claim 9 in sex identification of Penaeus vannamei, characterized in that: In the step S1, the weight of each of the Penaeus vannamei shrimp to be tested is not less than 2 g.

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