SNP (Single Nucleotide Polymorphism) site combination for detecting family survival rate in litopenaeus vannamei mixed culture process, molecular probe, kit and application

Abstract

The invention provides an SNP site combination for detecting the family survival rate in the litopenaeus vannamei mixed culture process, a molecular probe, a kit and application, and belongs to the technical field of litopenaeus vannamei culture. The molecular marker comprises 10416 SNP loci, and the physical positions of the SNP loci are determined based on comparison of a litopenaeus vannamei reference genome sequence. The invention further provides a molecular probe for capturing the SNP site combination, a kit comprising the molecular probe and application of the kit to DNA allele frequency acquisition and family survival rate detection in the litopenaeus vannamei mixed culture process. According to the method, the DNA allele frequency is obtained through the SNP site combination, the family survival rate of the litopenaeus vannamei in the mixed culture process is detected by calculating the contribution rate of each family of the litopenaeus vannamei in the mixed culture process, accurate evaluation of the family survival rate is achieved, the detection efficiency is improved, the detection cost is reduced, and use is convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Litopenaeus vannamei culture, in particular to a SNP site combination and a molecular probe for detecting the survival rate of a family in the mixed culture of Litopenaeus vannamei, a kit and an application. BACKGROUND

[0002] Litopenaeus vannamei, also known as white shrimp, is the highest single output value of marine aquaculture species in China and the world. In 2024, the total output of Litopenaeus vannamei in China reached 237.39 million tons, accounting for about 40% of the world's total output. Excellent shrimp is the source of high-quality development of the industry. So far, there have been 15 new varieties of Litopenaeus vannamei. The successful cultivation of these new varieties has laid a solid foundation for the self-sufficiency of Litopenaeus vannamei broodstock in China.

[0003] The selection breeding system of Litopenaeus vannamei is based on families, and traditional physical markers are used to carry out family mixed culture testing and pedigree tracking, and then the survival rate, disease resistance or environmental stress resistance of different families are selected. With the continuous increase of labor cost, the disadvantages of this method are increasingly prominent: first, the workload of physical markers is huge (more than 40,000 markers per generation), time-consuming, and it is difficult to carry out multi-trait and multi-environment testing at the same time due to cost and time constraints, which restricts the breeding progress of multi-trait excellent new varieties; second, the family needs to be temporarily raised for 2-3 months to reach the physical marker specification, which not only occupies a large amount of aquaculture facilities and affects the growth rate, but also introduces common environmental effects in the process of genetic evaluation, reducing the accuracy of breeding value estimation.

[0004] Molecular marker technology can break through the limitations of conventional breeding and enable target trait selection without being limited by gene expression and environmental conditions. SNP (Single nucleotide polymorphisms) markers are ideal molecular markers due to their rich genetic variation, widespread genomic distribution, and ease of automated typing. Some methods for identifying the relationship and detecting the survival rate of Litopenaeus vannamei using site combinations in mixed culture have appeared in the prior art. However, this detection method cannot accurately distinguish different families in mixed DNA sequencing typing due to the low heterozygosity of the site combination, and cannot achieve effective application of mixed DNA sequencing method. Moreover, this detection method requires sequencing each individual to analyze the survival rate of the family, which is complicated and costly. SUMMARY

[0005] The application aims to provide a SNP site combination for detecting the survival rate of a family in the mixed culture process of Litopenaeus vannamei, a molecular probe, a kit and an application, and aims to solve the problem that the method for identifying the genetic relationship and detecting the survival rate of Litopenaeus vannamei in the mixed culture process by using a site combination in the prior art cannot accurately distinguish different families in mixed DNA sequencing typing due to the low heterozygosity of the site combination, resulting in inaccurate survival rate detection results, and the operation is complicated and the detection cost is high because the method needs to perform sequencing typing on each individual in the detection process.

[0006] In order to solve the above technical problems, the technical scheme of the application is implemented as follows.

[0007] The SNP site combination for detecting the survival rate of a family in the mixed culture process of Litopenaeus vannamei comprises 10416 SNP sites, the physical position of the SNP sites is determined based on sequence alignment of a Litopenaeus vannamei reference genome, 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 Litopenaeus 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 Litopenaeus vannamei reference genome of the site.

[0008] Table 1 Information of SNP sites

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] The SNP locus combination for detecting family survival rate in mixed culture of Litopenaeus vannamei in this invention includes 10,416 SNP loci. This SNP locus combination has high heterozygosity and is used to obtain the allele frequencies of mixed DNA in families and culture populations during mixed culture, achieving high-precision detection of mixed DNA. This allows for accurate calculation of the contribution rate of different families in surviving individuals, rapid detection of family survival rate in mixed culture of Litopenaeus vannamei, and thus obtaining accurate survival rate data. This improves the accuracy of family survival rate detection in mixed culture of Litopenaeus vannamei and achieves precise assessment of family survival rate. Moreover, this SNP locus combination is a set of loci that is compatible with the mixed DNA sequencing method. Each family does not need to undergo physical labeling or individual sequencing genotyping, effectively reducing the common environmental effects caused by individual family culture. The method is simple, easy to operate, improves detection efficiency, and greatly reduces detection costs. The reference genome sequence version number of Litopenaeus vannamei in this invention is GCA_003789085.1, and the URL to obtain it is: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_003789085.1 / .

[0054] This invention discloses a molecular probe for detecting family survival rate during mixed farming of Litopenaeus vannamei. The molecular probe is a single-stranded DNA probe, which is designed based on 1-4 differentially expressed nucleotide sequences according to the SNP sites described in the SNP site combination for detecting family survival rate during mixed farming of Litopenaeus vannamei. Each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP site.

[0055] The molecular probes of this invention are molecular probes for detecting SNP site combinations in Litopenaeus vannamei. These molecular probe combinations can target 10416 regions. Single-stranded nucleotides of 110 bp in length are synthesized based on the designed nucleotide sequence. The molecular probes are DNA nucleotide sequences modified with a biotin group at the 5' end. The synthesized molecular probes are mixed in equimolar amounts using a mixture of EDTA and Tris-HCl to prepare a probe mixture. EDTA, ethylenediaminetetraacetic acid, has the chemical formula C6H2O. 10 H 16 N₂O₈; Tris-HCl, Tris (hydroxymethyl) Aminomethane Hydrochloride, with the molecular formula C₄H₂O. 12 ClNO3. DNA (deoxyribonucleic acid) is a base pair, a pair of matching bases in DNA and RNA linked together by hydrogen bonds. It is the basic unit for measuring the length of DNA and RNA.

[0056] The present invention discloses a kit for detecting family survival rate in the mixed culture of Litopenaeus vannamei, the kit comprising a probe mixture and a hybridization capture reagent, wherein the probe mixture is prepared from the molecular probe for detecting family survival rate in the mixed culture of Litopenaeus vannamei as described above.

[0057] The kit for detecting family survival rate in mixed culture of Litopenaeus vannamei of the present invention consists of individually packaged probe mixtures and hybridization capture reagents. The probe mixtures are prepared from molecular probes. The hybridization capture reagents are typically composed of Block I (blocker I), Block II (blocker II), 2× Hyb Buffer (2x hybridization buffer), HybBuffer Enhancer (hybridization buffer enhancer), Primer Mix (primer mixture), PCR Master Mix (PCR master mix), 2× Beads Wash Buffer (2x magnetic bead wash buffer), 10× Wash Buffer I (10x wash buffer I), 10× Wash Buffer II (10x wash buffer II), 10× Wash Buffer III (10x wash buffer III), 10× Strigent Wash Buffer (10x strigent wash buffer), DNA Probe Beads (DNA probe magnetic beads), and DNA Clean Beads (DNA purification magnetic beads). These hybridization capture reagents are preferably sourced from Shijiazhuang Borui Biotechnology Co., Ltd. This kit is low in cost and has good accuracy.

[0058] The present invention relates to the application of a kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in DNA allele frequency acquisition, comprising the following steps: 1) The DNA of the sample to be tested is broken, the ends are repaired, the adapters are ligated, and the DNA is purified to obtain a sequencing library. Then, the library is amplified and purified to obtain purified library DNA. 2) Add the probe mixture and hybridization capture reagent from the kit for detecting family survival rate during mixed culture of Litopenaeus vannamei as described above to the purified library DNA obtained in step 1) to perform hybridization capture, elution, and removal of DNA fragments in non-target regions to obtain target DNA fragments including the SNP sites. Amplify and purify the obtained target DNA to construct a targeted sequencing library. 3) The targeted sequencing library obtained in step 2) is tested. After passing the test, DNA sequencing is performed using next-generation sequencing technology. The sequencing results are compared with the reference genome sequence of Litopenaeus vannamei. Based on the number of reads covering REF and ALT bases, the allele frequencies of the sample to be tested at all sites are obtained.

[0059] The kit for detecting family survival rate in mixed culture of Litopenaeus vannamei of this invention can be used to obtain the allele frequency of DNA, especially the allele frequency of mixed DNA. This is a Litopenaeus vannamei mixed DNA genotyping method based on targeted capture sequencing. It utilizes the molecular probe of this invention to directionally capture target regions, which are DNA fragments including target sites, thereby obtaining the target DNA fragments. After DNA sequencing, the sequence is compared with the Litopenaeus vannamei reference genome sequence to obtain the allele frequency of the mixed DNA of the Litopenaeus vannamei to be tested. The SNP site combinations of this invention have high heterozygosity (0.2-0.6) and a large minimum allele frequency (MAF), with an average MAF of 0.2. It demonstrates good accuracy in assessing the contribution rate of mixed DNA to family analysis, with a detection rate of 99.79%. Simultaneously, it has advantages such as low detection cost and good site consistency between different sequencing batches, facilitating comparison and evaluation of tests across different batches. This method, which involves mixing DNA samples from multiple individuals and combining them with quantitative genotyping to estimate the frequency of each genetic marker locus in the mixed sample, can significantly reduce the number of genotyping samples and drastically lower the cost of genotyping. The overall sequencing cost is approximately 1 / 30th of that of traditional sequencing, or even lower. Furthermore, early mixed-breeding can eliminate the environmental effects of individual family rearing and improve the accuracy of genetic assessment.

[0060] In a preferred embodiment, in step 3), the DNA sequencing depth is not less than 10× / tail. In this invention, the DNA sequencing depth is not less than 10× / tail; for example, if DNA from 30 tails is mixed for sequencing, the sequencing depth is not less than 300×.

[0061] The present invention discloses a kit for detecting family survival rate in the polyculture of Litopenaeus vannamei, which is applied to the detection of family survival rate in the polyculture of Litopenaeus vannamei, comprising the following steps: S1 Preparations 1) Select individuals from different test families, with no fewer than 2 test families and no fewer than 60 individuals in each test family; S2 mixed farming 2) In each test family in step 1), 30-50 sibling individuals were randomly selected and placed in a parallel culture pond. They were then cultured together at a water temperature of 15-35℃ to obtain surviving individuals. S3 obtains allele frequencies 3) Take the paternal or maternal parent or sibling individuals of the test family in step 1) as target individuals, and use the steps described above for the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in DNA allele frequency acquisition to obtain the allele frequencies of all loci of the target individuals as the initial allele frequencies of the test family. 4) Count the number of surviving individuals obtained in step 2) and randomly divide them into several test groups. Mix the DNA of each individual in the group in equal amounts to obtain mixed DNA. Use the steps described above for the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in obtaining allele frequencies of mixed DNA to obtain the allele frequencies of the test group at all loci. S4 calculates survival rate 5) Among the allele frequencies of the test group obtained in step 4) and the initial allele frequencies of the test family obtained in step 3), let the test family have a total of n indivual( n ≥2), Indicates the first m Allele frequencies at each locus in the test group Indicates the first m Each locus in the test family i Initial allele frequencies in ( i =1, ..., n ), Indicates the first i The percentage of each tested family in the test group ( i =1, ..., n Construct the overdetermined system of equations shown in equation (I) to calculate the results for each test family in the test group. The contribution rate of each tested family in the test group was obtained; 6) Based on the family contribution rate obtained in step 5), add up the number of individuals in each test family in the test group, count the total number of surviving test individuals and the initial number of individuals in each test family, divide the number of surviving individuals in each test family by the initial number of individuals in each test family, and thus obtain the survival rate of different Litopenaeus vannamei families in the mixed culture process.

[0062] The kit for detecting family survival rate in mixed culture of Litopenaeus vannamei of this invention detects the family survival rate of Litopenaeus vannamei during mixed culture by evaluating the contribution rate of each family in a mixed population. It is a method for detecting family survival rate in mixed culture of Litopenaeus vannamei. First, the allele frequencies of the mixed DNA of families and the mixed culture population are obtained using SNP locus combinations. Then, by constructing an overdetermined system of equations, the contribution rate of each family in the mixed population is accurately calculated, thereby obtaining the survival rate data of each family. This method for detecting the survival rate of Litopenaeus vannamei families in mixed culture does not require individual rearing of individual families. Moreover, due to the rich genetic variation and large MAF of this SNP locus combination, the contribution rate of each family in the mixed population can be accurately obtained based on mixed DNA sequencing, replacing the traditional VIE marker. This effectively reduces the common environmental effects caused by individual rearing of families, improves the accuracy of detecting the survival rate of Litopenaeus vannamei families in mixed culture, and is easy to operate, highly efficient, and significantly reduces the detection cost, enabling efficient detection of family survival rates in mixed culture.

[0063] As a preferred embodiment, in step 3), during the process of obtaining the initial allele frequencies of the test family: if both parents of the test family are successfully genotyped, the initial allele frequencies of the test family are obtained using the genotyping information of the parents; if one or both parents of the test family fail to genotype, 30-50 sibling individuals are selected from the test family for mixed DNA genotyping, thereby obtaining the initial allele frequencies of the test family. In this invention, when establishing a family, sometimes both parents can be obtained; if samples from both parents are not complete, sibling individuals are used as substitutes, and the initial allele frequencies are obtained through mixed DNA sequencing. This invention provides two methods for obtaining the initial allele frequencies of the test family: one is parental (single-tail sequencing), and the other is sibling individuals (mixed DNA sequencing).

[0064] In a preferred embodiment, in step 4), the number of surviving individuals in each group is 30-50. In step 4), if the number of surviving individuals exceeds 60, they can be grouped into groups of 30-50. DNA is extracted from individuals within each group and mixed in equal amounts. The contribution rate of each family within each group is calculated, and then the contribution rates of each family in all groups are summed to calculate the overall survival rate.

[0065] As a preferred embodiment, the family survival rate refers to the family survival rate during normal mixed culture, the family survival rate with disease resistance during mixed culture, and the family survival rate with stress resistance during mixed culture. The method of the present invention for detecting the family survival rate of Litopenaeus vannamei during mixed culture can detect the family survival rate during normal mixed culture, as well as the family survival rate with disease resistance and the family survival rate with stress resistance during mixed culture. Normal mixed culture refers to culture where the shrimp grows naturally without external intervention; the family survival rate with disease resistance during mixed culture involves artificially introducing pathogens to simulate the disease-resistant growth process of Litopenaeus vannamei, also known as disease-resistant mixed culture; the family survival rate with stress resistance during mixed culture involves artificially intervening in the environment to create stress, simulating the stress-resistant growth process of Litopenaeus vannamei, also known as stress-resistant mixed culture. The survival rate detected by this invention can be the survival rate during natural growth, the survival rate when attacked by external pathogens, or the survival rate under the influence of the external environment, thus covering a wide range of detection.

[0066] In a preferred embodiment, in step 2), the normal mixed culture period is 30-90 days, the disease-resistant mixed culture period is 7-20 days, and the stress-resistant mixed culture period is 7-20 days. When testing the survival rate of families during normal mixed culture, the families are typically cultured together at a water temperature of 23-30℃ for 30-90 days; when testing the survival rate of disease-resistant families during mixed culture, the families are typically cultured together at a water temperature of 23-30℃ for 7-20 days; and when testing the survival rate of stress-resistant families during mixed culture, the families are typically cultured together at a water temperature of 15-35℃ for 7-20 days.

[0067] Compared with the prior art, the beneficial effects of the present invention are as follows: The SNP locus combination for detecting the survival rate of families in the mixed culture of Litopenaeus vannamei includes 10416 SNP loci. This SNP locus combination has high heterozygosity, and by using it to obtain the DNA allele frequencies of families and culture populations in the mixed culture process, high-precision DNA detection is achieved, especially high-precision detection of mixed DNA. This allows for accurate calculation of the contribution rate of different families in surviving individuals, rapid detection of the survival rate of Litopenaeus vannamei families in the mixed culture process, and thus obtaining accurate survival rate data, improving the accuracy of the survival rate detection of Litopenaeus vannamei families in the mixed culture process. Moreover, this SNP locus combination is a set of loci that is compatible with the mixed DNA sequencing method. Each family does not need to undergo physical labeling or individual sequencing genotyping, effectively reducing the common environmental effects caused by individual family culture. The method is simple, easy to operate, improves detection efficiency, and greatly reduces detection costs. Attached Figure Description

[0068] Figure 1 The flowchart illustrates the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei provided in this invention in detecting family survival rate in mixed culture of Litopenaeus vannamei. Figure 2 This is a scatter plot showing the predicted and actual survival rates of Litopenaeus vannamei tested in mixed culture of test families obtained in Embodiment 2 of the present invention. Detailed Implementation

[0069] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] This invention discloses a combination of SNP loci for detecting family survival rate during mixed culture of Litopenaeus vannamei. The combination of SNP loci includes 10,416 SNP loci. The physical location of the SNP loci is determined based on the alignment of the Litopenaeus vannamei reference genome sequence. The information of the SNP loci is shown in Table 1, where ID is the locus number, Scaffold is the Scaffold name of the Litopenaeus vannamei reference genome where the locus is located, Position is the physical location of the locus on the Scaffold, and Ref is the base name of the Litopenaeus vannamei reference genome where the locus is located.

[0071] This invention discloses a molecular probe for detecting family survival rate during mixed farming of Litopenaeus vannamei. The molecular probe is a single-stranded DNA probe, which is designed based on 1-4 differentially expressed nucleotide sequences according to the SNP sites described in the SNP site combination for detecting family survival rate during mixed farming of Litopenaeus vannamei. Each nucleotide sequence is 110 bp, and the average coverage range is 55 bp upstream and downstream of the SNP site.

[0072] The present invention discloses a kit for detecting family survival rate in the mixed culture of Litopenaeus vannamei, the kit comprising a probe mixture and a hybridization capture reagent, wherein the probe mixture is prepared from the molecular probe for detecting family survival rate in the mixed culture of Litopenaeus vannamei as described above.

[0073] The present invention relates to a kit for detecting family survival rate in mixed culture of Litopenaeus vannamei, and its application in obtaining allele frequencies in mixed DNA, comprising the following steps: 1) The DNA of the sample to be tested is broken, the ends are repaired, the adapters are ligated, and the DNA is purified to obtain a sequencing library. Then, the library is amplified and purified to obtain purified library DNA. 2) Add the probe mixture and hybridization capture reagent from the kit for detecting family survival rate during mixed culture of Litopenaeus vannamei as described above to the purified library DNA obtained in step 1) to perform hybridization capture, elution, and removal of DNA fragments in non-target regions to obtain target DNA fragments including the SNP sites. Amplify and purify the obtained target DNA to construct a targeted sequencing library. 3) The targeted sequencing library obtained in step 2) is tested. After passing the test, DNA sequencing is performed using next-generation sequencing technology. The sequencing results are compared with the reference genome sequence of Litopenaeus vannamei. The allele frequency of the sample at this site is obtained based on the number of reads covering REF and ALT bases.

[0074] Preferably, in step 3), the DNA sequencing depth is not less than 10× / tail.

[0075] The present invention discloses a kit for detecting family survival rate in the polyculture of Litopenaeus vannamei, which is applied to the detection of family survival rate in the polyculture of Litopenaeus vannamei, comprising the following steps: S1 Preparations 1) Select individuals from different test families, with no fewer than 2 test families and no fewer than 60 individuals in each test family; S2 mixed farming 2) In each test family in step 1), 30-50 sibling individuals were randomly selected and placed in a parallel culture pond. They were then cultured together at a water temperature of 15-35℃ to obtain surviving individuals. S3 obtains allele frequencies 3) Take the paternal or maternal parent or sibling individuals of the test family in step 1) as target individuals, and use the steps described above for the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in DNA allele frequency acquisition to obtain the allele frequencies of all loci of the target individuals as the initial allele frequencies of the test family. 4) Count the number of surviving individuals obtained in step 2) and randomly divide them into several test groups. Mix the DNA of each individual in the group in equal amounts to obtain mixed DNA. Use the steps described above for the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in DNA allele frequency acquisition to obtain the allele frequency of the test group at all loci. S4 calculates survival rate 5) Among the allele frequencies of the test group obtained in step 4) and the initial allele frequencies of the test family obtained in step 3), let the test family have a total of n indivual( n ≥2), Indicates the first m Allele frequencies at each locus in the test group Indicates the first m Each locus in the test family i Initial allele frequencies in ( i =1, ..., n ), Indicates the first i The percentage of each tested family in the test group ( i =1, ..., n Construct the overdetermined system of equations shown in equation (I) to calculate the results for each test family in the test group. The contribution rate of each tested family in the test group was obtained; 6) Based on the family contribution rate obtained in step 5), add up the number of individuals in each test family in the test group, count the total number of surviving test individuals and the initial number of individuals in each test family, divide the number of surviving individuals in each test family by the initial number of individuals in each test family, and thus obtain the survival rate of different Litopenaeus vannamei families in the mixed culture process.

[0076] Preferably, in step 3), during the process of obtaining the initial allele frequency of the test family: if the parents of the test family are successfully genotyped, the initial allele frequency of the test family is obtained using the genotyping information of the parents; if one individual from the parents of the test family is not successfully genotyped or neither parent is successfully genotyped, 30-50 sibling individuals are selected from the test family for mixed DNA genotyping, thereby obtaining the initial allele frequency of the test family.

[0077] Furthermore, in step 4), the number of surviving individuals in each group is 30-50.

[0078] Preferably, the family survival rate is the family survival rate during normal mixed farming, the family survival rate with disease resistance during mixed farming, and the family survival rate with stress resistance during mixed farming.

[0079] Furthermore, in step 2), the normal mixed breeding time is 30-90 days, the disease-resistant mixed breeding time is 7-20 days, and the stress-resistant mixed breeding time is 7-20 days.

[0080] Example 1

[0081] The present invention relates to a kit for detecting family survival rate in mixed culture of Litopenaeus vannamei, and its application in obtaining DNA allele frequencies, comprising the following steps: 1) Extraction and mixed-pond detection of Litopenaeus vannamei genomic DNA Muscle, swimming legs, or antennae tissue of Litopenaeus vannamei were selected, and DNA was extracted using a high-throughput DNA kit. The extracted DNA samples were subjected to two tests: first, the purity and integrity of the DNA were analyzed using 1% agarose gel electrophoresis; second, the concentration of the DNA was accurately quantified using a Qubit fluorescence quantitative analyzer.

[0082] 2) Construction of DNA library The DNA of the sample to be tested was randomly physically broken using an ultrasonic disruptor. The peak value of the broken fragments was controlled at 200-300bp, the ends were repaired, and the A tails were ligated. The DNA fragments with added A were ligated together with sequencing adapters using ligase, and the fragments were purified and selected using carboxyl-modified magnetic beads, retaining ligation products with insert fragments of 200-300 bp. Sequencing primers with barcodes and a high-fidelity PCR (Polymerase Chain Reaction) reaction system were added to the ligation product for PCR amplification. Different barcodes were used to distinguish different DNA samples. The DNA was purified using carboxyl magnetic beads to obtain the purified library.

[0083] 3) Construction of targeted sequencing libraries Take 500 ng of purified library DNA, freeze dry it, add the probe mixture and hybridization capture reagent from the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei of this invention, denature it, and incubate it at 65°C for 2 h to complete the hybridization capture reaction; The hybridization capture product was washed with a washing solution to remove DNA fragments from non-target regions and obtain the target DNA fragment. The obtained target DNA fragments were amplified and purified to construct a targeted sequencing library.

[0084] 4) DNA sequencing After the targeted sequencing library is constructed, it is first preliminarily quantified using a Qubit 2.0 fluorescence quantitative instrument, and then the effective concentration of the targeted sequencing library is accurately quantified using qPCR (Quantitative Real-time Polymerase Chain Reaction) to ensure that the obtained targeted sequencing library passes the test and thus guarantee the quality of the library. After the targeted sequencing library passes the testing, DNA sequencing is performed using next-generation sequencing technology.

[0085] 5) Genotype analysis Data quality control (removal of connectors and low-quality data), alignment with the Litopenaeus vannamei reference genome sequence, and variant detection.

[0086] 6) Calculation of allele frequencies Based on the number of reads covering REF and ALT bases, the allele frequencies of the sample at all loci were obtained.

[0087] Example 2

[0088] See appendix Figure 1 The present invention discloses a kit for detecting family survival rate in the polyculture of Litopenaeus vannamei, which is used in the detection of family survival rate in the polyculture of Litopenaeus vannamei, comprising the following steps: S1 Preparations 1) Select 60 families from the core breeding population of Litopenaeus vannamei.

[0089] S2 mixed farming 2) From each family in step 1), randomly select 30 juvenile shrimp at growth stage P15 and place them in one culture pond for mixed culture at a density of 110 shrimp / m². 3 The water temperature was 28℃, and the time was 70 days to obtain surviving test individuals.

[0090] S3 obtains allele frequencies 3) Take 30 sibling individuals from each family as target individuals for calculating the initial allele frequencies of the family. Use the method described in Example 1 to obtain the genotype information of the target individuals, i.e., the initial allele frequencies of the test family. 4) The number of surviving individuals in the culture pond was counted, and a total of 1232 individuals were harvested. The surviving individuals in the culture pond were divided into groups of 50, and a total of 15 groups were formed. DNA was extracted from each individual in each group and mixed in equal amounts, resulting in a total of 15 mixed DNA groups. The allele frequencies of the mixed groups at all loci were obtained using the method described in Example 1, which is the allele frequencies of the test groups.

[0091] S4 calculates survival rate In the allele frequencies of the test group obtained in step 4) and the initial allele frequencies of the test family obtained in step 3), let the test family have a total of n indivual( n ≥2), Indicates the first m Allele frequencies at each locus in the test group Indicates the first m Each locus in the test familyi Initial allele frequencies in ( i =1, ..., n ), Indicates the first i The percentage of each tested family in the test group ( i =1, ..., n Construct the overdetermined system of equations shown in equation (I) to calculate the results for each test family in the test group. The contribution rate of each tested family in the test group was obtained; 6) Based on the family contribution rate obtained in step 5), add up the number of individuals in each test family in the test group, count the total number of surviving test individuals and the initial number of individuals in each test family, divide the number of surviving individuals in each test family by the initial number of individuals in each test family, and thus obtain the survival rate of different Litopenaeus vannamei families in the mixed culture process.

[0092] From the appendix Figure 2 It can be seen that the number of surviving individuals in the Litopenaeus vannamei family obtained by the method of the present invention during the mixed culture process is highly consistent with the actual number of surviving individuals in the Litopenaeus vannamei family during the mixed culture process; therefore, the method of the present invention accurately obtains the family survival rate of Litopenaeus vannamei during the mixed culture process.

[0093] Therefore, compared with the prior art, the beneficial effects of the present invention are as follows: The SNP locus combination for detecting the survival rate of families in the mixed culture of Litopenaeus vannamei includes 10416 SNP loci. This SNP locus combination has high heterozygosity, and by using it to obtain the DNA allele frequencies of families and cultured populations in the mixed culture process, high-precision DNA detection is achieved, especially high-precision detection of mixed DNA. This allows for accurate calculation of the contribution rate of different families in surviving individuals, rapid detection of the survival rate of Litopenaeus vannamei families in the mixed culture process, and thus obtaining accurate survival rate data, improving the accuracy of the survival rate detection of Litopenaeus vannamei families in the mixed culture process. Moreover, this SNP locus combination is a set of loci that is compatible with the mixed DNA sequencing method. Each family does not need to undergo physical labeling or individual sequencing genotyping, effectively reducing the common environmental effects caused by individual family culture. The method is simple, easy to operate, improves detection efficiency, and greatly reduces detection costs.

[0094] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A combination of SNP loci for detecting family survival rate during mixed culture of Litopenaeus vannamei, characterized in that: The SNP locus combination includes 10,416 SNP loci, the physical locations of which were determined based on the alignment of the Litopenaeus vannamei reference genome sequence.

2. A molecular probe for detecting family survival rate during mixed culture of Litopenaeus vannamei, characterized in that: The molecular probe is a DNA single-stranded probe. The molecular probe is designed from 1 to 4 differentially expressed nucleotide sequences based on the SNP sites described in the SNP site combination for detecting family survival rate in the mixed culture of Litopenaeus vannamei according to claim 1. 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 detecting family survival rate during mixed culture of Litopenaeus vannamei, characterized in that: The kit includes a probe mixture and a hybridization capture reagent, wherein the probe mixture is prepared from a molecular probe for detecting family survival rate during mixed culture of Litopenaeus vannamei according to claim 2.

4. The application of a kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in obtaining DNA allele frequencies, characterized in that... Includes the following steps: 1) The DNA of the sample to be tested is broken, the ends are repaired, the adapters are ligated, and the DNA is purified to obtain a sequencing library. Then, the library is amplified and purified to obtain purified library DNA. 2) Add the probe mixture and hybridization capture reagent from the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei as described in claim 3 to the purified library DNA obtained in step 1) to perform hybridization capture, elution, removal of DNA fragments in non-target regions, obtain target DNA fragments including the SNP sites, amplify and purify the obtained target DNA, and construct a targeted sequencing library. 3) The targeted sequencing library obtained in step 2) is tested. After passing the test, DNA sequencing is performed using next-generation sequencing technology. The sequencing results are compared with the reference genome sequence of Litopenaeus vannamei. Based on the number of reads covering REF and ALT bases, the allele frequencies of the sample to be tested at all sites are obtained.

5. The application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei according to claim 4 in obtaining DNA allele frequencies, characterized in that: In step 3), the DNA sequencing depth is not less than 10× / tail.

6. A kit for detecting family survival rate in mixed culture of Litopenaeus vannamei, characterized in that, Includes the following steps: S1 Preparations 1) Select individuals from different test families, with no fewer than 2 test families and no fewer than 60 individuals in each test family; S2 mixed farming 2) In each test family in step 1), 30-50 sibling individuals were randomly selected and placed in a parallel culture pond. They were then cultured together at a water temperature of 15-35℃ to obtain surviving individuals. S3 obtains allele frequencies 3) Take the paternal or maternal parent or sibling individuals of the test family in step 1) as target individuals, and use the steps described in the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei according to claim 4 or 5 in the acquisition of DNA allele frequencies to obtain the allele frequencies of all loci of the target individuals as the initial allele frequencies of the test family. 4) Count the number of surviving individuals obtained in step 2) and randomly divide them into several test groups. Mix the DNA of each surviving individual in the group in equal amounts to obtain mixed DNA. Use the steps described in the application of the kit for detecting family survival rate in mixed culture of Litopenaeus vannamei in DNA allele frequency acquisition according to claim 4 or 5 to obtain the allele frequency of the test group at all loci, which is used as the allele frequency of the test group. S4 calculates survival rate 5) Among the allele frequencies of the test group obtained in step 4) and the initial allele frequencies of the test family obtained in step 3), let the test family have a total of n indivual( n ≥2), Indicates the first m Allele frequencies at each locus in the test group Indicates the first m Each locus in the test family i Initial allele frequencies in ( i =1, ..., n ), Indicates the first i The percentage of each tested family in the test group ( i =1, ..., n Construct the overdetermined system of equations shown in equation (I) to calculate the results for each test family in the test group. The contribution rate of each tested family in the test group was obtained; 6) Based on the family contribution rate obtained in step 5), add up the number of individuals in each test family in the test group, count the total number of surviving test individuals and the initial number of individuals in each test family, divide the number of surviving individuals in each test family by the initial number of individuals in each test family, and thus obtain the survival rate of different Litopenaeus vannamei families in the mixed culture process.

7. The application of the kit for detecting family survival rate in the polyculture of Litopenaeus vannamei according to claim 6 in the detection of family survival rate in the polyculture of Litopenaeus vannamei, characterized in that: In step 3), during the process of obtaining the initial allele frequencies of the test family: if the parents of the test family are successfully genotyped, the initial allele frequencies of the test family are obtained using the genotyping information of the parents; if one individual from the parents of the test family is not successfully genotyped or neither parent is successfully genotyped, 30-50 sibling individuals are selected from the test family for mixed DNA genotyping, thereby obtaining the initial allele frequencies of the test family.

8. The application of the kit for detecting family survival rate in the polyculture of Litopenaeus vannamei according to claim 6 in the detection of family survival rate in the polyculture of Litopenaeus vannamei, characterized in that: In step 4), the number of surviving individuals in each group is 30-50.

9. The application of the kit for detecting family survival rate in the polyculture of Litopenaeus vannamei according to claim 6 in the detection of family survival rate in the polyculture of Litopenaeus vannamei, characterized in that: The family survival rates are: family survival rate during normal mixed farming, family survival rate with disease resistance during mixed farming, and family survival rate with stress resistance during mixed farming.

10. The application of the kit for detecting family survival rate in the polyculture of Litopenaeus vannamei according to claim 9 in the detection of family survival rate in the polyculture of Litopenaeus vannamei, characterized in that: In step 2), the normal mixed breeding time is 30-90 days, the disease-resistant mixed breeding time is 7-20 days, and the stress-resistant mixed breeding time is 7-20 days.

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