Exopalaemon carinicauda 80K liquid phase chip, probe combination and application thereof

Abstract

The invention relates to an exopalaemon carinicauda 80K liquid phase chip, a probe combination and application of the exopalaemon carinicauda 80K liquid phase chip and the probe combination, and belongs to the technical field of crustacean genetic breeding. The SNP locus combination is composed of 81,097 SNP molecular markers, the position and base information of each SNP locus on the chip are as shown in a table 1, and the SNP loci are physically positioned on a genome GCF036898095.1. The chip sites are uniformly distributed on genome chromosomes, the coverage degree is high, the polymorphism is good, the universality is high, and the chip contains sites obviously related to growth, reproduction and saline-alkaline tolerance characters, and can be used for exopalaemon carinicauda population genetic diversity analysis, breeding population genotype identification, character correlation analysis and genome selective breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic breeding of crustaceans, and relates to the technical field of whole genome selection breeding, in particular to an 80K liquid chip for Exopalaemon carinicauda, a probe combination and application thereof. BACKGROUND

[0002] Exopalaemon carinicauda belongs to Palaemonidae and Palaemon. In recent years, as a new breeding variety, it has developed rapidly and become a major characteristic aquaculture variety in coastal mudflat areas of Jiangsu, Zhejiang and Shandong provinces. At present, two new varieties of Exopalaemon carinicauda, "Huangyu No. 1" and "Kesu No. 1", have been bred. However, with the innovation of breeding mode and the development of breeding scale, the existing new varieties of Exopalaemon carinicauda cannot meet the actual needs of the development of the breeding industry, and new varieties of Exopalaemon carinicauda with superior traits for different breeding environments are urgently needed to support the healthy development of the Exopalaemon carinicauda industry.

[0003] At present, the breeding of new varieties of Exopalaemon carinicauda mainly relies on traditional methods such as population selection. Although conventional breeding methods such as population breeding and family breeding can obtain aquatic products with excellent economic traits, these methods have limitations such as long breeding cycle, low breeding efficiency, poor predictability, and seriously restrict the further development of the aquatic species industry. Compared with traditional selection methods, whole genome selection breeding technology can effectively improve the accuracy of individual trait prediction and significantly reduce the cost of breeding. The economic traits (such as growth and stress resistance) of aquatic animals are usually controlled by micro-effect genes, and the low density of traditional molecular markers makes it difficult to accurately locate at the gene level. Molecular markers in the whole genome range can provide high-resolution positioning information and provide more accurate candidate targets for molecular breeding and genetic improvement. In recent years, with the rapid development of high-throughput sequencing technology, the whole genome maps of many important economic shrimps such as Litopenaeus vannamei, Marsupenaeus japonicus, Macrobrachium rosenbergii and Exopalaemon carinicauda have been analyzed. The continuous enrichment of aquatic organism genome information has laid a solid foundation for the application of whole genome selection breeding technology. Whole genome selection breeding technology has become one of the key technologies for breeding new aquatic varieties with excellent growth, stress resistance and other traits.

[0004] As a high-throughput genotyping platform developed after traditional SNP detection methods, SNP chip technology has become a core tool for modern biological breeding. Common gene chips mainly include solid-phase chips and liquid-phase chips. The solid-phase chip is permanently fixed on the carrier once designed, cannot be added or deleted, has poor flexibility, and has high single detection cost due to complex manufacturing process. Compared with the solid-phase chip, the liquid-phase chip based on targeted capture sequencing technology has probes existing in solution, can flexibly add, reduce or adjust sites by mixing different probe pools, has the characteristics of customized flexibility and high cost performance, has lower detection cost, higher throughput and simpler data analysis, and therefore has higher practicability in large-scale commercial breeding population screening, and has become the mainstream choice for promoting the application of genomic selection technology. The technology can detect tens of thousands of genetic markers at one time, realize precise, rapid and early evaluation of the genetic value of parent shrimps. By combining with the genomic selection strategy, the SNP chip technology can break the excessive dependence on phenotype data, predict the breeding value of multiple economic traits such as growth, stress resistance and quality of individuals at an early stage of life, greatly improve the selection accuracy and efficiency, and shorten the breeding period. The Exopalaemon modestus is an important economic shrimp in China, and its genome is large, so the cost of genotyping by using traditional resequencing technology is high, and therefore, a medium-low density liquid-phase chip needs to be developed for the whole genome selection breeding, whole genome association analysis, genetic diversity analysis and the like of the Exopalaemon modestus. SUMMARY

[0005] To solve the above problems, the application provides an Exopalaemon modestus 80K liquid-phase chip, a probe combination and application, the chip site is uniformly distributed on the genome chromosome, has high coverage, good polymorphism and strong universality, and the chip contains sites significantly related to growth, reproduction and salt-tolerant traits, and can be used for genetic diversity analysis of the Exopalaemon modestus population, genotype identification of the breeding population, trait association analysis and genomic selection breeding.

[0006] To achieve the above application purposes, the application is realized by the following technical solutions.

[0007] The application first provides an Exopalaemon modestus 80K liquid-phase chip, which comprises a probe corresponding to a SNP site combination; the SNP site combination is composed of 81,097 SNP molecular markers, the position and base information of each SNP site on the chip are shown in Table 1, and the SNP site is physically located on the genome GCF_036898095.1.

[0008] Further, the SNP site combination comprises 2,716 sites related to economic traits, including 547 growth trait-associated SNP sites, 1,498 reproduction trait-associated SNP sites, 530 salt-tolerance trait-associated SNP sites and 141 sex-associated SNP sites.

[0009] The application further provides a probe combination for identifying the genotypes of the SNP sites in the SNP site combination, which is used for identifying the 81,097 SNP molecular markers.

[0010] The application further provides applications of the 80K liquid chip for Pacifastacus leniusculus and the probe combination in Pacifastacus leniusculus genotyping.

[0011] Further, the application method comprises: extracting the genomic DNA of the Pacifastacus leniusculus sample to be detected; detecting the genomic DNA by using the liquid chip to obtain raw data; and performing data analysis on the raw data to obtain a genotyping result.

[0012] The application further discloses applications of the liquid chip and the probe combination in Pacifastacus leniusculus population genetic structure analysis, genetic breeding, genetic diversity analysis and whole genome association analysis.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The 80K liquid chip for Pacifastacus leniusculus provided by the application can quickly and accurately detect the genotypes of Pacifastacus leniusculus individuals, the average SNP detection rate is 97.8%, the genotyping stability is good, the accuracy is high, and the overall performance is excellent.

[0015] The 80K liquid chip provided by the application comprises significant sites related to growth, reproduction and salt tolerance. The whole genome 80K liquid chip for Pacifastacus leniusculus prepared by using the application can quickly genotype the Pacifastacus leniusculus sample, a small amount of sample can be detected, and there is no sample quantity limitation. The chip can be widely used in precise identification and evaluation of Pacifastacus leniusculus germplasm resources and varieties, construction of high-density genetic maps, QTL positioning, key gene mining, whole genome selection breeding, and provides important technical support for Pacifastacus leniusculus elite breeding, and can effectively improve the efficiency and precision of Pacifastacus leniusculus elite breeding.

[0016] The whole genome 80K liquid chip of Exopalaemon modestus provided by the application has higher flexibility compared with traditional solid-phase chips, can adjust the SNP density of the chip according to requirements, provides various versions such as 50K, 40K, 30K and 20K, and can capture sequence information of 100bp upstream and downstream of the target SNP site, provides more data for functional gene research, and adapts to different scale research and application requirements. The liquid chip can complete high-throughput detection, has higher detection flexibility, can effectively shorten the sample detection period, and improves the research efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The SNP marker chromosome distribution diagram of the 80K liquid chip of Exopalaemon modestus;

[0018] Figure 2 The SNP marker chromosome distribution statistical diagram of the 80K liquid chip of Exopalaemon modestus;

[0019] Figure 3 The Gap distribution diagram of the 80K liquid chip of Exopalaemon modestus;

[0020] Figure 4 The MAF distribution statistical diagram of the 80K liquid chip of Exopalaemon modestus;

[0021] Figure 5 The SNP marker type statistical diagram of the 80K liquid chip of Exopalaemon modestus;

[0022] Figure 6 The Manhattan diagram of the whole genome correlation analysis of the body length trait of Exopalaemon modestus;

[0023] Figure 7 The QQ diagram of the whole genome correlation analysis of the body length trait of Exopalaemon modestus;

[0024] Figure 8 The Manhattan diagram of the whole genome correlation analysis of the body weight trait of Exopalaemon modestus;

[0025] Figure 9 The QQ diagram of the whole genome correlation analysis of the body weight trait of Exopalaemon modestus. DETAILED DESCRIPTION

[0026] In the following examples, the 80K liquid chip of Exopalaemon modestus is developed based on a targeted capture technology, and the working principle is that target fragments are captured by designing probes based on target region sequences, high-throughput sequencing is carried out by using a sequencing platform, target genes and sites are detected, a large number of samples are quickly detected, and the purpose of target region gene detection and typing is achieved.

[0027] In the following examples of the present application, the liquid phase chip of Exopalaemon modestus 80K comprises a group of Exopalaemon modestus 80K SNP probe sites, and the 81,097 SNP sites are detected by the SNP probe sites.

[0028] In the following examples of the present application, the liquid phase chip of Exopalaemon modestus 80K comprises an independently packaged 80K probe mixture and a detection kit suitable for liquid phase hybridization capture.

[0029] In the following examples of the present application, the liquid phase chip of Exopalaemon modestus 80K is designed based on the principle that the probe interacts with the target sequence to combine, and the length of the probe is 110 bp.

[0030] In order to further illustrate the present application, the following describes in detail a liquid phase chip of Exopalaemon modestus 80K, a probe combination and an application thereof provided by the present application in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0031] Example 1: Design and preparation of the liquid phase chip of Exopalaemon modestus 80K

[0032] 1. Resequencing of population DNA samples and SNP calling

[0033] The DNA samples of 90 Exopalaemon modestus were resequenced (individual sequencing depth was 10x) and processed in combination to obtain 1,070,946 SNP sites, i.e. original SNP sites. The 90 Exopalaemon modestus were respectively from 10 Exopalaemon modestus EC5 inbred lines, 10 EC4 inbred lines, 10 closed colony populations, 10 Zhanjiang populations, 10 salt-tolerant families, 10 Rizhao wild populations, 10 Kosu Red No. 1, and 20 Huangyu No. 1 populations.

[0034] 2. SNP screening and quality control

[0035] The BWA software (parameters: mem-t 4-k 32-M) was used to align to the Exopalaemon modestus reference genome (accession number GCF_036898095.1), and the alignment results were removed by SAMTOOLS (parameter: rmdup). It was ensured that the original SNP sites were consistent with the target reference genome, and 18,161,339 SNP sites consistent with the target reference genome were obtained.

[0036] 3. Preparation of the liquid phase breeding chip

[0037] The functional SNP sites that can successfully design the probes were screened from the original SNP sites: the screening process included site filtering and site evaluation in turn.

[0038] (1) Selection principle of candidate sites: heterozygosity ≤ 50%, deletion rate ≤ 20%, MAF ≥ 0.1, uniform distribution, and meeting the site evaluation principle, and designing the probe;

[0039] (2) Site evaluation principle: Extract the upstream and downstream sequences of the SNP site, and follow the probe length of 110 bp, GC content of 30-70%, and the number of homologous regions ≤5;

[0040] According to the principle of uniform distribution, the sites of the successfully designed probes are selected, and then 81,097 SNP sites are selected as the final site set according to the capture stability, wherein the core sites are 2,716 (547 growth trait associated sites, 1,498 reproduction trait associated sites, 530 salt-tolerant trait associated sites, and 141 sex associated sites).

[0041] The SNP marker chromosome distribution map based on the above-mentioned 81,097 SNP sites is shown in FIG. 1; the SNP marker chromosome distribution statistical map based on the above-mentioned 81,097 SNP sites is shown in FIG. 2; the Gap distribution map based on the above-mentioned 81,097 SNP sites is shown in FIG. 3; the MAF distribution statistical map based on the above-mentioned 81,097 SNP sites is shown in FIG. 4; and the SNP marker type statistical map based on the above-mentioned 81,097 SNP sites is shown in FIG. 5. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

[0042] Example 2: Method for obtaining genotype data of Exopalaemon modestus by 80K liquid chip

[0043] 1. DNA extraction and quality evaluation of the Exopalaemon modestus sample to be detected

[0044] Randomly select 300 Exopalaemon modestus samples for 80K liquid chip detection, and use a high-throughput DNA extraction kit to extract the DNA of the Exopalaemon modestus sample to be detected. The DNA sample integrity is detected by agarose gel electrophoresis with a quality fraction of 1%, and the main band of the sample is clear and complete according to the gel imaging system detection; the genomic DNA concentration is detected by Qubit 2.0 spectrophotometer, and the DNA concentration is guaranteed to be ≥100 ng / μL, 1.8≤OD 260 / OD 280 ≤2.0, and OD 260 / OD 230 ≥2.0.

[0045] 2. Obtain genotype data based on the Exopalaemon modestus 80K liquid chip

[0046] ​​​​​DNA treated with restriction enzyme fragmentation was end-repaired and A-tailed, and then ligated with barcode adapters and purified by carboxyl-modified magnetic beads. The product was amplified by high-fidelity PCR and purified twice. 500 ng of the purified library was vacuum-dried and hybridized with GenoBaits probes at 65°C for two hours. Non-specific fragments were removed by washing, and the captured library was constructed by PCR. Finally, the library concentration was quantified using a Qubit 2.0 spectrophotometer, and PE150 sequencing was performed on the MGISEQ-T7 platform.

[0047] The raw sequencing data was trimmed for adapter sequences and low-quality reads using fastp, and the obtained high-quality reads were aligned to the reference genome using BWA-MEM under default parameters. PCR duplicates were removed using Picard Tools to reduce amplification bias, and SNP sites were detected using the Genome Analysis Toolkit (GATK) following the GATK best practice workflow. Finally, SNP genotype data for 300 individuals of Exopalaemon carinicauda was successfully obtained for candidate population genetics analysis.

[0048] Example 3 Application of Exopalaemon carinicauda 80K Liquid Chip in Genome-wide Association Analysis

[0049] Genotype data for 816 Exopalaemon carinicauda samples was obtained using the Exopalaemon carinicauda 80K liquid chip prepared in Example 1. Genome-wide association analysis was performed on body length and body weight traits of Exopalaemon carinicauda.

[0050] The unified expression of the genome-wide association analysis model is: y = Xa + Qb + Km + e

[0051] Where y is the phenotype vector, X is the genotype matrix, a is the genotype effect vector, Q is the fixed effect matrix (which can be population structure / gender / location / occasion, etc.), b is the fixed effect vector, K is the random effect matrix, mainly the kinship matrix, m is the random effect vector, and e is the residual error vector. For each SNP site, it is tested whether a is 0. The probability value p of a being 0 is used to measure the degree of association between marker genotype and phenotype. The smaller the p value, the smaller the probability of a being 0, and the more likely the marker is associated with the trait.

[0052] The calculation of the commonly used model GLM of GWAS was realized by using Tassel software. The population structure matrix corresponding to the optimal K value of admixture was used as the Q matrix of the corresponding model, and the kinship matrix between samples calculated by gcta software was used as the K matrix of the corresponding model. The obtained p value was taken-log10, and then the Manhattan plot and Q-Q plot were shown. In the 816 Exopalaemon modestus populations, 40 SNP sites related to body length traits and 118 SNP sites related to body weight traits were further screened, which provided basic data for molecular marker assisted selection breeding of Exopalaemon modestus growth traits.

[0053] The Manhattan plot and QQ plot drawn based on the whole genome association analysis of 81,097 SNP sites and body length traits are shown in Figures 1 and 2. Figure 6 and Figure 7 The Manhattan plot and QQ plot drawn based on the whole genome association analysis of 81,097 SNP sites and body weight traits are shown in Figures 3 and 4. Figure 8 and Figure 9 .

[0054] The Manhattan plot shows the degree of association between each SNP and the trait, and the dashed line in the figure corresponds to the significance threshold. According to the Q-Q plot, it can be observed that the distribution of scatter points basically coincides with the diagonal line. From the horizontal coordinate greater than 2, the P value of the GWAS result has a significant gap with the uniformly distributed P value, indicating that there is indeed a significant correlation between the phenotype and the genotype.

[0055] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

[0056] Table 1: Position of SNP site

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Claims

1. A liquid chip of Exopalaemon modestus 80K, characterized in that, The chip comprises a probe corresponding to a SNP site combination; the SNP site combination comprises 81,097 SNP molecular markers, the position and base information of each SNP site on the chip are shown in Table 1, and the SNP site is physically located on the genome GCF_036898095.

1.

2. The liquid chip of Exopalaemon modestus 80K according to claim 1, wherein, The SNP site combination comprises 2,716 sites related to economic traits, wherein 547 SNP sites are associated with growth traits, 1,498 SNP sites are associated with reproduction traits, 530 SNP sites are associated with salt-tolerant traits, and 141 SNP sites are associated with gender.

3. A combination of probes for discriminating the genotypes of each SNP site, characterized in that, The probe combination is used for identifying the 81,097 SNP molecular markers in the 80K liquid chip of Exopalaemon modestus in claim 1.

4. Application of the 80K liquid chip of Exopalaemon modestus in claim 1 or the probe combination in claim 3 in genotyping of Exopalaemon modestus.

5. Use according to claim 4, characterized in that, The method of the application comprises: extracting genomic DNA of a sample of Exopalaemon modestus to be detected; detecting the genomic DNA by using the liquid chip to obtain raw data; and performing data analysis on the raw data to obtain a genotyping result.

6. Application of the liquid chip in claim 1 or the probe combination in claim 3 in population genetic structure analysis, genetic breeding, genetic diversity analysis and whole genome association analysis of Exopalaemon modestus.

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