Genome-wide SNP molecular marker combination, liquid phase microarray and its application in the ovate pomfret

Abstract

The invention discloses a SNP molecular marker combination for whole genome selection of silver pomfret, wherein the SNP molecular marker combination comprises any combination of 40475 SNP sites, and information of the 40475 SNP sites is specifically shown in Table 1. The invention also discloses a whole genome liquid phase chip and a kit of silver pomfret based on the SNP molecular marker combination, and the SNP molecular marker combination, the kit or the SNP liquid phase chip can be used in whole genome association analysis of silver pomfret, genotyping of silver pomfret, breeding or assisted breeding of silver pomfret, and detection of DNA samples of silver pomfret. The invention develops a SNP chip specifically for silver pomfret, which is of great significance for improving the utilization efficiency of germplasm resources, accelerating the breeding process of excellent traits, and enhancing the economic value and environmental adaptability of aquaculture varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of genomics, and specifically relates to a whole genome SNP molecular marker combination, a liquid phase chip and applications of an oval pomfret, and in particular to a whole genome about 40K breeding liquid phase chip of an oval pomfret based on liquid phase probe capture targeted sequencing genotyping technology (Genotyping By Target Sequencing, GBTS) and its application. Background Art

[0002] As one of the important aquatic species in deep-sea aquaculture in my country, the aquaculture output of the oval pomfret has increased significantly in recent years, which is of great significance to the development of the aquaculture industry. However, in the traditional aquaculture process, due to the lack of efficient and precise genetic improvement technology, the aquaculture of the oval pomfret faces challenges such as low efficiency of germplasm resource utilization, weak stress resistance and disease resistance. These problems limit the sustainable development and production increase of the oval pomfret industry.

[0003] With the development of molecular biology, functional genomics and bioinformatics, genomic breeding technology has gradually become the key to solving these problems. In particular, single nucleotide polymorphism (SNP) as a genetic marker has shown extremely high application value in genetic diversity analysis, gene positioning, trait association research and molecular assisted selection breeding. However, the research and development of high-throughput and high-efficiency SNP chip technology for oval pomfret is still in its infancy.

[0004] Some existing aquaculture species have begun to apply genomic breeding technology and carry out efficient genetic improvement through SNP chips. The successful application of these technologies shows that the development of SNP chips specifically for oval pomfret is of great significance for improving the utilization efficiency of germplasm resources, accelerating the breeding process of excellent traits, and enhancing the economic value and environmental adaptability of farmed varieties. Therefore, the development of a dedicated genomic SNP chip for oval pomfret is not only an important demand in the field of genetic breeding of oval pomfret, but also a key step in promoting scientific and technological innovation and sustainable development in the aquaculture industry. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker combination and a kit for whole genome selection of oval pomfret.

[0006] The present invention also aims to provide a liquid phase chip of the whole genome of oval pomfret and a preparation method thereof.

[0007] The last object of the present invention is to provide the application of the above-mentioned SNP molecular marker combination, liquid phase chip or kit.

[0008] The first object of the present invention can be achieved by the following technical scheme: a SNP molecular marker combination for whole genome selection of oval pomfret, the SNP molecular marker combination comprising any combination of 40475 SNP sites, and the information of the 40475 SNP sites is specifically shown in Table 1 below:

[0009] Table 1 Information of 40475 SNP loci

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[0117] ; The physical locations of the 40,475 SNP sites were determined by sequence alignment based on the reference genome of the oval pomfret with version number GCA_900607315.1.

[0118] The reference genome can also refer to Zhang DC, Guo L, Guo HY, et al. Chromosome-level genome assembly of golden pompano (Trachinotus ovatus) in the family Carangidae. Sci Data. 2019; 6(1): 216. Published 2019 Oct 22. doi: 10.1038 / s41597-019-0238-8.

[0119] Preferably, the 40,475 SNP sites described in the present invention include 34 SNP sites related to economic traits of oval pomfret and 40,441 background SNP sites, and the SNP sites related to economic traits include 21 SNP sites related to growth traits, 5 SNP sites related to hypoxia resistance, 5 SNP sites related to resistance to cryptonucleosis, and 3 SNP sites related to gender.

[0120] The present invention also provides a kit, which comprises probes and / or primers for detecting the SNP molecular marker combination.

[0121] The second object of the present invention can be achieved by the following technical solution: a liquid phase chip of the whole genome of oval pomfret, the liquid phase chip comprising probes and / or primers for detecting the SNP molecular marker combination.

[0122] Preferably, the method for preparing the oval pomfret whole genome liquid phase chip comprises the following steps:

[0123] (1) Screening high-quality SNP sites from the resequencing data of the oval pomfret;

[0124] (2) SNP loci related to hypoxia tolerance, resistance to Cryptocaryon irritans, body weight, morphological traits, sex, and background SNP loci were screened from the genome-wide association analysis data of the oval pomfret;

[0125] (3) removing the SNP sites for which probes cannot be designed at both ends from the SNP sites;

[0126] (4) Search for SNP intervals longer than 2000 bp and add SNPs that may cover important genes, repeating the process multiple times until no sites can be filled;

[0127] (5) The SNP sites are grouped together to obtain 40,475 SNP sites, and probes and / or primers are designed according to the SNP sites to obtain a liquid phase chip of the whole genome of the oval pomfret.

[0128] The third object of the present invention can be achieved by the following technical solution: application of the above-mentioned SNP molecular marker combination, kit or SNP liquid phase chip in genome-wide association analysis of oval pomfret.

[0129] The present invention obtains 34 SNP sites associated with target economic traits through whole genome association analysis and SNP screening in relevant candidate genes, including hypoxia resistance, cryptorchidism resistance, body weight, morphological traits, gender, etc.

[0130] The present invention also provides the use of the above-mentioned SNP molecular marker combination, kit or SNP liquid phase chip in genotyping of oval pomfret.

[0131] The present invention also provides the use of the above-mentioned SNP molecular marker combination, kit or SNP liquid phase chip in the breeding or assisted breeding of oval pomfret.

[0132] The present invention also provides the use of the above-mentioned SNP molecular marker combination, kit or SNP liquid phase chip in detecting a DNA sample of an oval pomfret.

[0133] Furthermore, the use of the above-mentioned SNP molecular marker combination, kit or SNP liquid phase chip in detecting a DNA sample of an oval pomfret comprises the following steps:

[0134] (1) Extraction of genomic DNA from oval pomfret: Genomic DNA was extracted from the fin tissue of oval pomfret using the CTAB method according to standard procedures;

[0135] (2) DNA quality detection: 1% agarose gel electrophoresis was used to detect the genomic DNA concentration using NanoDrop to ensure that the DNA concentration was ≥10 ng / μL and 1.8 ≤ OD 260 / OD 280 ≤2.0, OD 260 / OD 230 ≥2.0, the integrity of the DNA sample was tested by gel imaging to ensure that the main band of the sample was clear and the integrity was good;

[0136] (3) Liquid phase chip detection: The sample is tested using the SNP liquid phase chip to obtain the genotype of the sample to be tested.

[0137] The invention has the following advantages: based on the SNP molecular marker combination for whole genome selection of oval pomfret, the invention develops a whole genome liquid phase chip and a kit for oval pomfret; the SNP molecular marker combination, the kit or the SNP liquid phase chip can be used in whole genome association analysis of oval pomfret, gene typing of oval pomfret, breeding or assisted breeding of oval pomfret, and detection of DNA samples of oval pomfret; the invention develops a SNP chip specifically for oval pomfret, which is of great significance for improving the utilization efficiency of germplasm resources, accelerating the breeding process of excellent traits, and enhancing the economic value and environmental adaptability of aquaculture varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 The chromosome distribution diagram (A) and distribution statistics diagram (B) of the SNP marker "Pomfish Core No. 1" provided in Example 1 of the present invention;

[0139] Figure 2 Example 1 of the present invention provides the distribution of the marker "Pomfish Core No. 1" in the gene structure (A) and the LD attenuation diagram based on the target site (B);

[0140] Figure 3 The site detection rate of the "Pomfish Core No. 1" whole genome breeding liquid phase chip provided in Example 2 of the present invention;

[0141] Figure 4 This is the site consistency rate of the "Pomfish Core No. 1" whole genome breeding liquid phase chip provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0142] Example 1

[0143] The following examples are used to illustrate the content of the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0144] The technical solution of the present invention is to promote the germplasm innovation and breeding of oval pomfret, and is assisted by Boridi Biotechnology Co., Ltd. Based on the resequencing VCF file and site information collection table of oval pomfret provided by the customer, the company conducts feasibility assessment and site verification, site evaluation and site selection, and designs and synthesizes a 45K site panel based on liquid probe captured targeted sequencing genotyping technology (Genotyping By Target Sequencing, GBTS) for testing, selects 40K sites that meet the requirements from the test results, and synthesizes a set of final usable oval pomfret panels, completing the development of the oval pomfret 40K marker detection kit.

[0145] Example 1

[0146] Acquisition of SNP molecular marker combination for whole genome selection of oval pomfret:

[0147] (1) Whole genome acquisition

[0148] Fin tissue of an oval pomfret was selected, DNA was extracted, and 105Gb of data was obtained by second-generation illunima sequencing after library construction, and 16.9Gb of Hifi data was obtained by sequencing on the PacBio platform. Hifiasm was used to quickly construct haplotypes-de novo assembly of PacBio Hifireads data, and second-generation data was used for correction. Finally, a genome with a total length of 647.5Mb was constructed, and contigN50 reached 1.80Mb.

[0149] (2) Acquisition of the first type of sites

[0150] Whole genome resequencing was performed on fin ray samples of 200 ovate pomfret populations from 10 major aquaculture areas, including Guangdong, Guangxi Zhuang Autonomous Region and Hainan (see Table 2), and 13,982,173 SNP markers were obtained. By integrating SNP loci related to growth, hypoxia tolerance, resistance to cryptorchidism and sex, 40,475 markers were finally selected to construct a liquid phase gene chip. The chip locus information is shown in Table 1 above.

[0151] SNP marker chromosome distribution diagram (A) and distribution statistics diagram (B) are shown in Figure 1 As shown in Figure 2, the distribution of SNP molecular markers in the gene structure (A) and the LD attenuation diagram based on the target site (B) are shown in Figure 2. Figure 2 shown.

[0152] Depend on Figure 1 and Figure 2It can be seen that the gene chip SNP markers are evenly distributed on the 24 chromosomes of the oval pomfret. The average number of SNP markers in each chromosome is 1686, ranging from 1022 to 2525. The vast majority of SNPs are located in the introns and intergenomic regions of the oval pomfret genome, and the markers have sufficient coverage of the entire genome.

[0153] Specifically, the Illumina PE 150 platform was used to perform whole genome resequencing on 200 wild oval pomfret (see Table 2) from ten different sea areas in China, with a total sequencing volume of 1480.84G and an average sequencing depth of 11.42× for each sample. The specific process is to construct a sequencing library for each sample, sequence all DNA libraries on the Illumina PE 150 platform, and filter out:

[0154] 1) Reads with low quality and adapter contamination, reads with low quality (the number of bases with quality value Q≤5 accounts for more than 50% of the entire read);

[0155] 2) Remove reads containing N (N means the base information cannot be determined) with a ratio greater than 5%;

[0156] 3) For reads with adapter contamination, all clean data were aligned to the genome sequence of the oval pomfret (version number is GCA_900607315.1) using BWA software, variant site detection and typing were performed using GATK software, and the retained variant sites were annotated using snpEff software, and finally 13,982,173 SNP sites were screened.

[0157] From the above SNP sites, we removed the SNP sites where high-quality DNA probes could not be designed on both sides, searched for SNP intervals exceeding 2000 bp, supplemented SNPs that might cover important genes, and repeated this process many times until no sites could be filled. We further screened SNP sites based on the principle of uniform distribution on the genome, and finally obtained a total of 40,475 high-quality SNP sites.

[0158] Table 2 Resequencing results of ten populations of Pisces ovalis

[0159] Sampling location Number of samples Average sequencing amount per sample / G Average sequencing depth / X Shenzhen 20 7.121 10.989 Zhuhai 20 8.815 13.603 Yangxi 20 6.976 10.765 Suixi 20 7.501 11.576 Xuwen 20 7.137 11.014 Lingshui 20 7.242 11.176 Changjiang 20 7.119 10.986 Lingao 20 7.661 11.823 North Sea 20 7.008 10.815 Qinzhou 20 7.462 11.515

[0160] (3) The second type of loci were obtained: 34 SNP loci associated with the target economic traits were obtained based on whole-genome association analysis and SNP screening within related candidate genes.

[0161] Specifically, taking the screening of SNP loci related to hypoxia tolerance as an example, 500 wild pomfret from Shenzhen were randomly selected, and the dissolved oxygen in the water was treated below the suffocation point. The death time of each fish was recorded as the basis for determining its hypoxia tolerance.

[0162] The fin rays of 50 oval pomfret that died first and last were cut, and DNA was extracted using a magnetic bead DNA extraction kit. 20 mg of fin rays were taken, and reagents were added in sequence according to the kit instructions to extract the DNA of the oval pomfret. The extracted DNA was stored in 150 μL Buffer AE, and the DNA samples were detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were detected by Nanodrop2000, requiring the A260 / A280 ratio to be between 1.8 and 2.0, and the DNA concentration to be greater than 100 μg / μL.

[0163] The genomic DNA was randomly broken into fragments of 350 bp in length. After end repair, phosphorylation and A-tailing, the two ends of the fragments were connected to adapters to prepare a DNA library, and then PE150 sequencing was performed on the Illumina platform. The whole-base group retesting method adopted in the present invention can mine rare variants, has a high SNP density, and can be applied to whole-genome association analysis;

[0164] The obtained genotype data were quality-controlled and filtered using PLINK (V1.07) software, with the following filtering conditions: detection rate > 95%, minor allele frequency (MAF) < 0.05, Hardy-Weinberg equilibrium test P < 10 -6 , finally 100 individuals and 3,377,860 SNPs were used for GWAS analysis;

[0165] GWAS association analysis was performed using mixed linear models (MLM) using GEMMA software.

[0166] y=Wα+Xβ+Zμ+ε

[0167] Among them, Y is the phenotypic value, that is, the survival time of oval pomfret in low dissolved oxygen, W is the covariance matrix, α is the overall mean, β is the effect of the SNP marker, and X is its indicator variable. Z is the kinship matrix based on SNPs, μ is the additive genetic effect, and ε is the random residual. The factors affecting the phenotype were subjected to principal component analysis, and the first five principal components were added to the model as covariates.

[0168] Five SNP molecular markers associated with the hypoxia tolerance trait of oval pomfret were discovered through genome-wide association analysis (GWAS).

[0169] The acquisition method of other SNP loci related to economic traits is similar.

[0170] The loci information of the finally obtained oval pomfret breeding gene chip “Pomfish Core 1” is shown in Table 3.

[0171] Table 3 Loci information of the gene chip “Pompano core 1” for breeding of oval pomfret

[0172] Key Parameters Parameter Value Marker Type SNP Total number of tags 40475 Growth trait related 21 Hypoxia tolerance traits 5 Correlation between Cryptocaryon irritans resistance and 5 Gender related 3 Background sites 40441

[0173] The physical positions of the 40,475 SNP sites were determined by sequence alignment based on the reference genome of the oval pomfret (version number: GCA_900607315.1).

[0174] Preparation of the "Pomfish Core No. 1" whole genome breeding liquid phase chip: Probes and / or primers were designed based on 40,475 SNP sites (Shijiazhuang Boridi Biotechnology Co., Ltd.) to obtain the oval pomfret whole genome liquid phase chip, named "Pomfish Core No. 1" whole genome breeding about 40k liquid phase chip.

[0175] A kit can also be prepared, which contains probes and / or primers for detecting the SNP molecular marker combination.

[0176] Example 2

[0177] Application of the whole genome breeding liquid phase chip of Pomfret core No. 1 in detecting DNA samples of oval pomfret and in genotyping of oval pomfret

[0178] The development and verification was carried out using the “Shenxin No. 1” egg-shaped pomfret breeding liquid phase chip:

[0179] (1) Extraction of genomic DNA from oval pomfret: Fin ray tissues of oval pomfret were dissected and sampled according to standard procedures, and genomic DNA was extracted using the CTAB method;

[0180] (2) DNA sample quality detection: Detection by 1% agarose gel electrophoresis; use UV spectrophotometer to detect genomic DNA concentration to ensure that the DNA concentration is ≥100 ng / μL and 1.8 ≤ OD 260 / OD 280 ≤2.0, OD 260 / OD 230 ≥2.0; Detect the integrity of the DNA sample using a gel imager to ensure that the main band of the sample is clear and has good integrity;

[0181] (3) Liquid phase chip testing: Through the standardized process operation and quality control of Huazhi Company, the samples are subjected to library construction, target region capture and high-throughput sequencing;

[0182] (4) Bioinformatics analysis: Freebayes software was used to analyze the variant sites of the sequencing results to obtain the genotype of the target site; IGV software was used to view the variations between the sequencing results and the reference genome.

[0183] Among the 163 samples of oval pomfret tested, the average detection rate of all loci was 99.94%, and the average consistency rate with the resequencing genotype was 96.23%. The above data show that the cGPS test results meet the development standards (see Figure 3 and Figure 4 ).

[0184] The SNP molecular marker combination, the kit or the SNP liquid phase chip is used to detect the oval pomfret DNA to be tested, and a suitable oval pomfret is selected for subsequent breeding.

[0185] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A SNP molecular marker combination for whole genome selection of ovate pomfret, Features: The SNP molecular marker combination includes 40475 SNP loci, and the information of the 40475 SNP loci is specifically shown in Table 1 below: Table 1 Information of 40475 SNP loci The physical positions of the 40,475 SNP sites were determined by sequence alignment based on the reference genome of the oval pomfret with version number GCA_900607315.

1.

2. The SNP molecular marker combination according to claim 1, Features: The 40,475 SNP loci include 34 SNP loci related to economic traits of oval pomfret and 40,441 background SNP loci. The SNP loci related to economic traits include 21 SNP loci related to growth traits, 5 SNP loci related to hypoxia tolerance, 5 SNP loci related to resistance to cryptonarianosis, and 3 SNP loci related to gender.

3. A kit, Features: The kit comprises probes and / or primers for detecting the SNP molecular marker combination according to claim 1.

4. A liquid phase microarray of the whole genome of the oval pomfret, Features: The liquid phase chip comprises probes and / or primers for detecting the SNP molecular marker combination according to claim 1.

5. The method for preparing the whole genome liquid phase chip of the oval pomfret according to claim 4, Features The following steps are involved: (1) Screening high-quality SNP sites from the resequencing data of the oval pomfret; (2) SNP loci related to hypoxia tolerance, resistance to Cryptocaryon irritans, body weight, morphological traits, sex, and background SNP loci were screened from the genome-wide association analysis data of the oval pomfret; (3) removing the SNP sites for which probes cannot be designed at both ends from the SNP sites; (4) Search for SNP intervals longer than 2000 bp and add SNPs that may cover important genes, repeating the process multiple times until no sites can be filled; (5) The SNP sites are grouped together to obtain 40,475 SNP sites, and probes and / or primers are designed according to the SNP sites to obtain a liquid phase chip of the whole genome of the oval pomfret.

6. Use of the SNP molecular marker combination according to claim 1 or 2, the kit according to claim 3 or the SNP liquid phase chip according to claim 4 in genome-wide association analysis of silver pomfret.

7. Use of the SNP molecular marker combination according to claim 1 or 2, the kit according to claim 3 or the SNP liquid phase chip according to claim 4 in genotyping of oval pomfret.

8. Use of the SNP molecular marker combination according to claim 1 or 2, the kit according to claim 3 or the SNP liquid phase chip according to claim 4 in breeding or assisted breeding of oval pomfret.

9. Use of the SNP molecular marker combination according to claim 1 or 2, the kit according to claim 3 or the SNP liquid phase chip according to claim 4 in detecting a DNA sample of oval pomfret.

10. The use according to claim 9, Features The following steps are involved: (1) Extraction of genomic DNA from oval pomfret: Genomic DNA was extracted from the fin tissue of oval pomfret using the CTAB method according to standard procedures; (2) DNA quality detection: 1% agarose gel electrophoresis was used to detect the genomic DNA concentration using NanoDrop to ensure that the DNA concentration was ≥10 ng / μL and 1.8 ≤ OD 260 / OD 280 ≤2.0, OD 260 / OD 230 ≥2.0, the integrity of the DNA sample was tested by gel imaging to ensure that the main band of the sample was clear and the integrity was good; (3) Liquid phase chip detection: The sample is tested using the SNP liquid phase chip to obtain the genotype of the sample to be tested.

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