Takifugu obscurus 20K liquid phase chip as well as construction method and application thereof

Abstract

The invention discloses a takifugu obscurus 20K liquid phase chip and a construction method and application thereof, the chip comprises 20017 SNP sites uniformly distributed on a takifugu obscurus chromosome, and 4129 SNP sites are associated with temperature, growth and sex traits. The Takifugu obscurus 20K liquid phase chip prepared by the invention has the advantages that the genotyping cost is greatly reduced, the genotype consistency rate and the site detection rate are extremely high, the Takifugu obscurus 20K liquid phase chip can be widely applied to the fields of Takifugu obscurus important economic character mining, QTL (quantitative trait loci) positioning, whole genome selective breeding and the like, and the accuracy and high efficiency of excellent germplasm breeding of Takifugu obscurus are expected to be improved.

Description

Technical Field

[0001] The invention relates to a Takifuge obscurus 20K liquid phase chip, a construction method and application thereof, and belongs to the field of aquatic products. Background Art

[0002] Agriculture is based on seeds. Good seeds are the "chips" of the aquaculture industry and the foundation for ensuring national food safety and ecological security. Good varieties are the core elements and lifeline of modern fisheries and the key to sustainable development of fisheries. With the development of biotechnology, breeding technology with genomic selection as the core has been deeply studied and widely used in the livestock and aquatic fields. Compared with other traditional breeding methods, genomic selection breeding has many advantages. For example, accurate genome-wide SNP markers can effectively improve the accuracy of selection; multiple traits can be selected at the same time, significantly improving the efficiency of selection; SNP genotypes can be tested in the juvenile period, greatly shortening the breeding cycle and saving costs; and the accuracy of selection of low-heritability traits can be significantly improved.

[0003] At present, there are two types of chips based on SNP technology, solid-phase chips and liquid-phase chips. Solid-phase chips use microarray technology to base complement the probe with the sequence to be tested, and through the design of probes and fluorescent markers, they can capture and obtain the genetic information of the site to be tested. Due to its high requirements for sample processing and the need to pre-treat the target molecule, the experimental operation is relatively complicated and the chip cost is relatively high. Liquid-phase chips are based on the complementary binding of target probes and target sequences for fixed-point capture. Liquid-phase chips have the advantages of small sample usage, simple and fast operation, high sensitivity and low cost. At present, a number of liquid-phase chips have been developed and applied in the fields of livestock and poultry and aquatic products, such as: domestic donkey 40K liquid-phase chip, dairy cow 85K liquid-phase chip, large yellow croaker 55K liquid-phase chip, largemouth black bass 100K liquid-phase chip, and vannamei 40K liquid-phase chip. Liquid-phase chips can be widely used in the fields of biological evolution, genetic map construction, marker association analysis, etc.

[0004] The dark-striped pufferfish, commonly known as pufferfish, is a precious migratory omnivorous fish that is loved by people for its delicious meat. With the expansion of the scale of dark-striped pufferfish farming, the germplasm resources have been degraded due to inbreeding and overfishing of wild resources. Specifically, the cold resistance and growth rate have decreased, which has seriously restricted the development of the dark-striped pufferfish farming industry. It is urgent to use genomic selection technology to cultivate new germplasm with faster growth rate, stronger disease resistance and better tolerance to low temperature environment. High-density SNP liquid phase chip is conducive to the improvement of excellent traits of dark-striped pufferfish, early selection, shortening the breeding cycle, and effectively improving the efficiency and accuracy of its breeding of fine varieties. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a combination of SNP sites in the whole genome of Takifugu obscurus. The second purpose of the present invention is to provide a Takifugu obscurus 20K liquid phase chip, its construction method and application.

[0006] Technical solution: The present invention provides a whole-genome SNP site combination of dark-spotted pufferfish, wherein the SNP site combination includes 20,017 SNP sites located on the chromosomes of dark-spotted pufferfish, including 15,888 background SNP sites and 4,129 functional SNP sites, wherein the functions include the cold resistance time, growth traits, and sex traits of dark-spotted pufferfish, the background SNP site information is shown in Table 1 in the specification, and the functional SNP site information is shown in Table 2 in the specification.

[0007] Table 1 Background SNP site information

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[0062] Table 2 Functional SNP site information

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[0078] The present invention also provides a Takifuge obscurus 20K liquid phase chip, wherein the liquid phase chip comprises probes corresponding to the Takifuge obscurus SNP site combination described in claim 1.

[0079] The present invention also provides a method for constructing the above-mentioned Takifugu obscurus 20K liquid phase chip, characterized in that the method comprises the following steps:

[0080] (1) Tail fin samples of dark-striped pufferfish from different geographical populations were collected and genomic DNA was extracted. The genome sequence of dark-striped pufferfish from Japan was obtained from the NCBI database, and the library was constructed and sequenced with the genomic DNA of the collected samples. The obtained data were quality controlled, genome compared, variant detected and filtered, and high-quality background SNP sites were selected.

[0081] (2) Subject individuals in the breeding population of dark-striped pufferfish to low temperature treatment, growth typing and sex classification to obtain phenotypic information of all individuals. At the same time, tail fin samples of all individuals were collected, genomic DNA was extracted, and library sequencing was performed. The obtained data were subjected to quality control, genome comparison, variation detection and filtering to obtain genotypes, and then SNP loci were screened to obtain SNP loci associated with the length of cold tolerance, growth traits, and sex traits;

[0082] (3) According to the site design requirements and probe design principles, capture probes are designed for the SNP sites obtained in steps (1) and (2), and finally a dark-striped pufferfish 20K liquid phase chip is synthesized.

[0083] Furthermore, the NCBI project name of the Japanese region dark-striped puffer genome sequence described in step (1) is PRJDB3165.

[0084] Furthermore, the filtering conditions in steps (1) and (2) are that the minimum allele frequency of SNP is ≥ 0.05, the missing rate of SNP is ≤ 0.01, the heterozygosity rate is ≤ 0.01, and the significance level of Hardy-Weinberg equilibrium is 0.00000001.

[0085] Furthermore, the different geographical groups mentioned in step (1) include Jiangsu and Guangdong, and the number of each group is no less than 6.

[0086] Furthermore, the temperature of the low temperature treatment in step (2) is 13°C.

[0087] Furthermore, the number of samples of the dark-striped pufferfish breeding population described in step (2) is 100.

[0088] Furthermore, the growth traits described in step (2) include body weight, total length, body length, body thickness, head length, snout length, caudal peduncle length, caudal peduncle height, interorbital width, eye diameter and body height.

[0089] The present invention also provides the application of the above-mentioned dark puffer whole genome SNP site combination and dark puffer 20K liquid phase chip in dark puffer genotyping, trait association analysis and whole genome selection breeding.

[0090] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the present invention discloses for the first time a 20K liquid phase chip for dark-striped pufferfish and its application, and the liquid phase chip is specific to dark-striped pufferfish. The present invention contains 20017 SNP loci combinations from dark-striped pufferfish populations in multiple regions, and contains a large number of loci associated with temperature, growth and sex traits, which can be applied to the molecular breeding of dark-striped pufferfish related traits, including but not limited to dark-striped pufferfish SNP breeding chip design, trait association analysis and whole genome selection breeding. In addition, the cost of the liquid phase chip genotyping developed by the present invention is greatly reduced, which is about 50% lower than that of traditional sequencing technology and solid phase chip, and the site detection rate is extremely high (greater than 97%), and the genotype consistency rate is extremely high (greater than 99%). This low-cost and high-efficiency genotyping method can greatly accelerate the molecular breeding process of dark-striped pufferfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Density map of the 20,017 SNP sites described in the present invention.

[0092] Figure 2 Detection rate of sites in 20K liquid microarray of Takifugu obscurus.

[0093] Figure 3 The genotype consistency rate of repeated samples of Takifugu obscurus 20K liquid phase array.

[0094] Figure 4 LOD distribution map of growth and cold-resistance traits of Takifugu obscurus. DETAILED DESCRIPTION

[0095] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0096] Example 1 Development of 20K Liquid Phase Breeding Chip for Takifugu obscurus

[0097] In this embodiment, by collecting caudal fin samples of Takifugu obscurus from different geographical populations and obtaining information from the NCBI database, and recording the temperature, growth, and cold-resistance traits of the candidate population, 20,017 SNP sites were screened out by a unique method after resequencing, and the Takifugu obscurus 20K breeding liquid phase chip was synthesized. The specific development steps are as follows:

[0098] (1) Fifteen tail fin samples of Takifugu obesum were collected from Jiangsu and Guangdong, respectively, and six genome sequences of Takifugu obesum from Japan were obtained from the NCBI database PRJDB3165 project;

[0099] Genomic DNA was extracted from all caudal fin samples (DNA extraction kit was from Nanjing Novezan, catalog number 017E2282GA) and library sequencing was performed (sequencing depth was 26X). Combined with the genomic data of the Japanese population, the obtained genotype data were subjected to quality control, genome alignment, variation detection and screening and filtering. The screening and filtering conditions used were that the minimum allele frequency of SNP was ≥ 0.05, the missing rate of SNP was ≤ 0.01, the heterozygosity rate was ≤ 0.01, and the significance level of Hardy-Weinberg equilibrium was 0.00000001. Finally, a total of 15,888 high-quality background SNP sites were selected, and the site information is shown in Table 1.

[0100] (2) 100 juvenile dark-striped pufferfish from the breeding population of Jiangsu Zhongyang Group were fed normally for 3 months, then subjected to low-temperature treatment (the dark-striped pufferfish were treated at 13°C and the time of fish body imbalance was recorded), growth typing (the following information was recorded: body weight, total length, body length, body thickness, head length, snout length, caudal peduncle length, caudal peduncle height, interorbital width, eye diameter and body height) and sex classification to obtain phenotypic information of all individuals, and caudal fin samples of all individuals were collected;

[0101] All tail fin samples were classified according to growth rate, cold tolerance time and sex, and then genomic DNA was extracted and library sequenced (sequencing depth was 26X). The obtained genotype data were subjected to quality control, genome alignment, variation detection and screening and filtering. The screening and filtering conditions used were that the minimum allele frequency of SNP was ≥ 0.05, the missing rate of SNP was ≤ 0.01, the heterozygosity rate was ≤ 0.01, and the significance level of Hardy-Weinberg equilibrium was 0.00000001. After SNP site screening, a total of 4129 SNP sites associated with cold tolerance time, growth traits and sex traits were obtained. The site information is shown in Table 2.

[0102] (3) The SNP sites obtained in (1) and (2) above were combined together, with a total of 20,017 SNP sites, as the final SNP site combination. The above SNP site combination was handed over to Huazhi Biotechnology Co., Ltd. for probe design and synthesis to obtain a 20K liquid-phase breeding chip.

[0103] The density diagram of the 20017 SNP sites in each chromosome of the dark-striped pufferfish is shown in Figure 1 .

[0104] Example 2 Quality Evaluation of Takifugu Obscurus 20K Liquid Chip Genotyping

[0105] A number of tail fin samples of Takifugu obscurus were selected from the preparatory population of Jiangsu Zhongyang Group, and genotyping detection and production testing were performed using the Takifugu obscurus 20K liquid phase chip developed in Example 1. The genotype consistency rate and site detection rate were extremely high, proving that the Takifugu obscurus 20K liquid phase chip developed by the present invention has an excellent effect. The specific test data are as follows:

[0106] (1) Demo test results

[0107] 10 samples of Takifugu obscurus were selected for genotyping using the Takifugu obscurus 20K liquid phase chip in Example 1. After sequencing and data analysis, the average detection rate of the Takifugu obscurus loci was 98.90%, and the genotype consistency rate with the resequencing samples was 99.34%. This demo test had a high genotype detection rate, good accuracy and stability, and excellent overall performance, which met the production test standards and could be used for production testing.

[0108] (2) Production test results

[0109] 90 new samples of Takifugu obscurus were selected for Takifugu obscurus 20K liquid phase chip production test. After sequencing and data analysis, the average detection rate of loci was 97.98% (e.g. Figure 2 The average genotype consistency of technical replicate samples was 99.07% (as shown in Figure 3 This production test has a high genotype detection rate, good accuracy and stability, and excellent overall performance, meeting development standards.

[0110] Example 3 Application of Takifugu obscurus 20K liquid phase chip in trait association analysis

[0111] (1) Determination of growth and cold tolerance of Takifugu obscurus: 152 Takifugu obscurus were selected and subjected to low temperature treatment (13°C). The time of fish body imbalance under low temperature was recorded in detail (as a criterion for cold tolerance), and growth-related traits such as body weight, body length, and total length were recorded. The tail fin tissue of the samples was also retained.

[0112] (2) Association analysis of growth and cold tolerance traits of Takifugu obscurus: DNA samples of the caudal fin of the above 152 Takifugu obscurus were extracted and a library was constructed. Targeted sequencing was performed using Takifugu obscurus 20K liquid phase chip. The sequencing data of 152 Takifugu obscurus were used for SNP detection and screening using the GATK protocol. A total of 185,990,380 SNPs were obtained, which were merged into 5520 polymorphic markers for association analysis. Using the Lep-Map3 software package with an LOD threshold of 11.0, 4891 markers (accounting for 88.6% of the 5520 polymorphic markers) were successfully located on 22 LGs. Four QTL positioning analyses were performed, including three growth traits and one cold tolerance-related trait (see Figure 4). The growth and cold-resistance traits are concentrated on LG2, LG15, LG19 and LG20, indicating that these linkage groups are very important for developing Takifugu obscurus germplasm with faster growth and better cold-resistance.

[0113] Example 4 Application of Takifugu obscurus 20K liquid phase chip in genomic selection breeding

[0114] 152 tail fin samples of Takifugu obscurus in Example 3 were collected, and genotyping was performed using Takifugu obscurus 20K liquid phase chip. DNA was extracted from the tail fin tissue of the above samples and sequenced to build a library. Analysis showed that the detection rate and genotype consistency rate of this batch of samples were high, both greater than 97%. ASReml software was used to predict and rank the genomic breeding values ​​of Takifugu obscurus samples, and the top-ranked samples were compared with samples with fast growth and good cold resistance. The comparison consistency was good, indicating that the Takifugu obscurus 20K liquid phase chip has high reliability in genomic selection breeding.

Claims

1. A whole genome SNP locus combination of Takifugu obscurus, characterized in that: The SNP site combination includes 20017 SNP sites located on the chromosomes of dark-striped pufferfish, including 15888 background SNP sites and 4129 functional SNP sites, and the functions include the cold resistance time, growth traits, and sex traits of dark-striped pufferfish. The background SNP site information is shown in Table 1 in the specification, and the functional SNP site information is shown in Table 2 in the specification.

2. A 20K liquid phase chip of Takifuge obscurus, characterized in that: The liquid phase chip comprises probes corresponding to the combination of SNP sites of Takifugu obscurus described in claim 1.

3. The method for constructing the Takifugu obscurus 20K liquid phase chip according to claim 2, characterized in that: The method comprises the following steps: (1) Tail fin samples of dark-striped pufferfish from different geographical populations were collected and genomic DNA was extracted. The genome sequence of dark-striped pufferfish from Japan was obtained from the NCBI database, and the library was constructed and sequenced with the genomic DNA of the collected samples. The obtained data were quality controlled, genome compared, variant detected and filtered, and high-quality background SNP sites were selected. (2) Subject individuals in the breeding population of dark-striped pufferfish to low temperature treatment, growth typing and sex classification to obtain phenotypic information of all individuals. At the same time, tail fin samples of all individuals were collected, genomic DNA was extracted, and library sequencing was performed. The obtained data were subjected to quality control, genome comparison, variation detection and filtering to obtain genotypes, and then SNP loci were screened to obtain SNP loci associated with the length of cold tolerance, growth traits, and sex traits; (3) According to the site design requirements and probe design principles, capture probes are designed for the SNP sites obtained in steps (1) and (2), and finally a dark-striped pufferfish 20K liquid phase chip is synthesized.

4. The construction method according to claim 3, characterized in that: The NCBI project name of the Japanese region dark-striped puffer genome sequence described in step (1) is PRJDB3165.

5. The construction method according to claim 3, characterized in that: The filtering condition in steps (1) and (2) is that the minimum allele frequency of the SNP is ≥ 0.05, the missing rate of SNP ≤ 0.01, the heterozygosity rate ≤ 0.01, and the significance level of Hardy-Weinberg equilibrium was 0.00000001.

6. The construction method according to claim 3, characterized in that: The different geographical groups mentioned in step (1) include Jiangsu and Guangdong, and the number of each group is no less than 6.

7. The construction method according to claim 3, characterized in that: The temperature of the low temperature treatment in step (2) is 13°C.

8. The construction method according to claim 3, characterized in that: The number of samples of the dark-striped pufferfish breeding population described in step (2) is 100.

9. The construction method according to claim 3, characterized in that: The growth traits described in step (2) include body weight, total length, body length, body thickness, head length, snout length, caudal peduncle length, caudal peduncle height, interorbital width, eye diameter and body height.

10. Use of the whole genome SNP locus combination of Takifugu obscurus described in claim 1 and the Takifugu obscurus 20K liquid phase chip described in claim 2 in genotyping, trait association analysis and whole genome selection breeding of Takifugu obscurus.

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