Special SNP chip for resisting visceral white-spot disease character of pseudosciaena crocea and application of special SNP chip

Abstract

The invention discloses an SNP chip special for the visceral white-spot disease resistance character of large yellow croaker and application of the SNP chip, the whole-genome SNP chip for the visceral white-spot disease resistance character of the large yellow croaker comprises a probe combination, and the probe combination is used for identifying the genotype of each SNP site in the SNP site combination; the large yellow croaker whole-genome SNP chip provided by the invention contains 44948 loci, 420 of the loci are key SNP loci for resisting visceral white-spot disease, the large yellow croaker whole-genome SNP chip can be applied to large yellow croaker disease-resistant character genome selective breeding, and by adopting the large yellow croaker whole-genome SNP chip disclosed by the invention, a large yellow croaker sample can be quickly subjected to genetic typing; the method is applied to selective breeding of a visceral white-spot disease resistant character genome, and is beneficial to shortening the breeding period of the large yellow croaker and accelerating the breeding process of a new variety of the large yellow croaker.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a Larimichthys crocea anti-visceral white spot disease trait specific SNP chip and application thereof. BACKGROUND

[0002] DNA molecular markers are an important basis for molecular breeding. Through molecular markers, genetic diversity analysis, genetic structure and germplasm resource identification of aquatic animals can be carried out, key functional genes in important aquatic animals can be screened, excellent germplasm can be quickly identified, and genetic improvement based on molecular marker assisted selection can be realized. Single nucleotide polymorphism (SNP) has the characteristics of large number, high detection accuracy, etc., and is the most commonly used molecular marker at present, which effectively promotes the transformation of aquatic animals from phenotype selection to genomic selection (GS). SNP genotyping detection technology mainly includes whole genome resequencing technology and SNP chip detection technology. The former can capture whole genome variations without preference, and is suitable for discovering new SNP sites, but has problems such as complex experimental process, large data volume (≥10 Gb per sample), high analysis cost, etc., which is difficult to meet the needs of large-scale breeding. In comparison, SNP chip is based on pre-defined sites to design probes, which realizes high-throughput, low-cost and accurate genotyping, and is widely used in livestock and aquatic animal breeding.

[0003] Larimichthys crocea belongs to Perciformes, Sciaenidae and Larimichthys, and is called "four major marine products" together with Pseudosciaena polyactis, Trichiurus haumela and Sepiella inermis, which has very important economic value. According to geographical distribution, Larimichthys crocea can be divided into three populations, namely Daiqu population, Minyue east population and Naozhou population, and there is certain difference in genome sequence information of different populations. Larimichthys crocea is an important cultured fish in China, and the disease problem is very serious in its cultivation process, among which the white spot disease caused by Pseudomonas plecoglossicida causes a large number of individual death and huge economic loss. Due to the relative lag of genetic improvement of disease resistance traits, there is a lack of disease-resistant fine varieties of Larimichthys crocea, which hinders the healthy and rapid development of Larimichthys crocea industrialization, and it is urgent to carry out fine variety breeding and obtain new varieties of Larimichthys crocea with excellent disease resistance traits.

[0004] Therefore, using efficient molecular breeding technology and method to quickly identify excellent germplasm and breed new varieties with excellent traits, realize the leap-forward development of genetic improvement and fine variety breeding of disease resistance traits of Larimichthys crocea, is an important way to promote the development of Larimichthys crocea culture. In order to improve the efficiency of Larimichthys crocea selection breeding, it is urgent to develop a Larimichthys crocea disease resistance trait whole genome SNP chip covering whole genome, high throughput, high accuracy and low cost, to meet the needs of Larimichthys crocea disease-resistant fine variety breeding. SUMMARY

[0005] Therefore, the application provides a Larimichthys crocea anti-visceral white spot disease trait special SNP chip and application thereof.

[0006] To solve the above technical problems, the application adopts the following technical scheme:

[0007] The Larimichthys crocea anti-visceral white spot disease trait special SNP chip comprises a probe combination, and the number of SNPs is 44948, and all SNP sites are located on a genome chromosome.

[0008] Preferably, the number of sites corresponding to the chromosome numbers of the chromosomes is respectively:

[0009] Chr01-Chr24: 2260, 1979, 1998, 1458, 2463, 1896, 2140, 2077, 1963, 1669, 1695, 2392, 1464, 2395, 1800, 1700, 1845, 1541, 2253, 1666, 2050, 1580, 1388, 1276.

[0010] Preferably, the genome is a Daiqu group Larimichthys crocea genome obtained by using PacBio SMRT and Hi-C technology.

[0011] Preferably, among the SNP sites, there are 420 anti-visceral white spot disease trait key SNP sites.

[0012] Preferably, the number of the 420 sites on each chromosome is respectively:

[0013] Chr01-Chr24: 29, 29, 4, 14, 20, 12, 12, 9, 19, 26, 22, 13, 11, 26, 14, 17, 4, 11, 54, 4, 17, 17, 26, 10.

[0014] Application of the SNP site combination in preparation of a Larimichthys crocea whole genome SNP chip.

[0015] Application of the SNP site combination in Larimichthys crocea genome selection breeding.

[0016] A Larimichthys crocea whole genome genotyping detection method comprises the following steps:

[0017] (1) Obtain the genomic DNA of a to-be-detected Larimichthys crocea sample;

[0018] (2) Detect the genomic DNA by using a Larimichthys crocea anti-disease trait whole genome SNP chip to obtain raw data;

[0019] (3) performing data analysis on the original data to obtain a genotyping result.

[0020] The present application has the following technical effects relative to the prior art:

[0021] The application provides a molecular marker combination for whole genome genotyping of large yellow croaker against internal viscera white spot disease, comprising 44948 SNP sites.

[0022] The application uses the above-mentioned molecular marker combination to construct a large yellow croaker genome selection breeding chip against internal viscera white spot disease, and the chip sites are uniformly distributed on the whole genome chromosome, and have high coverage.

[0023] The large yellow croaker whole genome SNP chip prepared by the application can quickly genotype large yellow croaker samples, can be applied to genome selection breeding of large yellow croaker against disease traits, and is helpful to shorten the breeding cycle of large yellow croaker and accelerate the breeding process of new varieties of large yellow croaker. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a SNP site detection rate diagram of the large yellow croaker genome selection breeding chip against internal viscera white spot disease of the application. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the application will be described below in the embodiments of the application with reference to the accompanying drawings, obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the application.

[0026] The application discloses a SNP chip special for large yellow croaker against internal viscera white spot disease, and the chip comprises a probe combination, the number of SNPs is 44948, and all the SNP sites are located on the genome chromosome.

[0027] The number of sites corresponding to the chromosome number of the chromosome is shown in Table 1:

[0028] Table 1:

[0029]

[0030] Among the SNP sites, 420 are key SNP sites for internal viscera white spot disease resistance, as shown in Table 2:

[0031] Table 2:

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] The number of 420 anti-internal white spot disease trait key SNP sites distributed on the chromosomes of the genome is shown in Table 3:

[0038] Table 3:

[0039]

[0040] The application also discloses application of the SNP site combination in preparation of a large yellow croaker whole genome SNP chip.

[0041] The application also discloses application of the SNP site combination in large yellow croaker genome selection breeding.

[0042] The application also discloses a large yellow croaker whole genome genotyping detection method, which comprises the following steps:

[0043] (1) obtaining genomic DNA of a large yellow croaker sample to be detected;

[0044] (2) detecting the genomic DNA by using a large yellow croaker disease resistance trait whole genome SNP chip to obtain original data;

[0045] (3) performing data analysis on the original data to obtain a genotyping result.

[0046] Example 1:

[0047] A high-quality genome sequence of the Daiqu group large yellow croaker is obtained by using PacBio SMRT and Hi-C technology, the genome size is 721.26 Mb, contig N50 = 28.48 Mb, scaffold N50 = 31.39 Mb, assembled into 24 chromosomes, the genome mounting rate is 97.86%, the genome sequence has been uploaded to NCBI (NCBI: SRR30870370), and the number of annotated genes is 25147. The specific implementation is as follows:

[0048] High-throughput sequencing and data quality control: The Illumina NovaSeq 6000 sequencing technology was used to perform paired-end sequencing on the sample DNA. The adapter sequences in the reads were removed using Trimmomatic-0.39 software, the 5' end of the reads containing non-AGCT bases was removed, the ends of the reads with low sequencing quality (sequencing quality value less than Q20) were trimmed, reads containing N with a proportion of 10% were removed, and small fragments with a length of less than 75 bp after adapter and quality trimming were removed. Finally, 40G of high-quality detection data was obtained. Meanwhile, 75G of high-quality Hi-C data was obtained. The Pacbio SMRT technology was used for sequencing, and the raw sequencing data was processed as follows: filter out Polymerase reads with a length of less than 200 bp; filter out Polymerase reads with a quality of less than 0.80; extract Subreads from Polymerase reads and filter out adapter sequences; filter out Subreads with a length of less than 500 bp. The filtered PacBio Subreads were 32G.

[0049] Genome assembly: The software Hifiasm was used to assemble the third-generation Pacbio SMRT sequencing data, and the genome size of large yellow croaker was 721.26G, and the scaffold N50 was 31.39Mb. The genome mounting rate was 97.86%. According to the sequencing Hi-C data, the software ALLHIC was used to mount the contigs / scaffolds sequence obtained by assembly to the near-chromosome level, and then the juicebox software was used to manually correct according to the strength of chromosome interaction, and finally the chromosome level genome was obtained, and the length of each chromosome is shown in Table 3.

[0050] Table 4:

[0051]

[0052]

[0053] Coding gene analysis: de novo prediction and homologous sequence alignment were combined to predict genes in the genome, and de novo gene prediction was performed by AUGUSTUS v3.2.3 software; homologous alignment prediction was performed using the protein sequence of the reference genome, the protein sequence was quickly aligned to the sample genome sequence (tblastn), the poor alignment results were filtered, and the redundancy was removed, and then Genewise v2.4.1 was run for accurate alignment to determine the coding region and intron region of the gene; the above gene set was integrated by EVidenceModeler v1.1.1 software, and 25147 coding genes of the sample genome were obtained.

[0054] Example 2:

[0055] Based on the genomic information obtained in Example 1, the genomic resequencing of 72 samples of wild populations and 1360 samples of cultured populations of P. major was performed, and the resequencing data was used for variation analysis to obtain large-scale whole genome variation information; high-quality SNP sites with population and individual representation were obtained by filtering and screening, combined with QTL positioning and GWAS analysis to screen SNP sites significantly related to important economic traits, and according to the genome annotation and variation annotation, the determination of the polymorphic site segment was performed, and finally the SNP chip was synthesized at the genome level. The specific implementation is as follows:

[0056] Based on the high-quality reference genome of P. major (NCBI: SRR30870370), the resequencing data of wild populations and cultured populations was used for variation analysis: after the DNA extracted from the tissue was qualified by agarose gel electrophoresis and Nanodrop detection, it was randomly broken into fragments with a length of 350bp by a Covaris crusher. The DNA fragments were end-repaired, poly A-tailed, adapter-ligated, purified, and PCR amplified to complete the whole library preparation. The constructed library was sequenced by illumina NovaSeq 6000, and the paired-end raw data obtained by sequencing was used to remove reads with adapters, reads with a proportion of N greater than 5%, and low-quality reads with a number of bases with a quality value Q<=10 accounting for more than 20% of the entire reads, to obtain high-quality effective data. The sequencing fragments were aligned back to the reference genome using BWA alignment software. According to the alignment results, GATK software was used to analyze the reference genome alignment, variation site screening and filtering of the filtered P. major wild and cultured population resequencing data, and high-quality SNP sites were obtained. ANNOVAR software was used to annotate the SNP detection results.

[0057] QTL mapping to screen trait-associated SNP sites: The genomic sequences of 152 samples of F1 full-sib offspring and parents were determined by genome resequencing technology, and SNP sites were screened by alignment with the reference genome to construct a high-density genetic linkage map. The linkage map contained 390,937 markers, with a map length of 1871.83 cM and was divided into 24 linkage groups. Combined with the phenotype information of the full-sib family resistance to internal white spot disease, 62 trait-associated SNP sites were successfully screened by QTL mapping analysis.

[0058] GWAS to screen trait-associated SNP sites: Genotype information of 1208 samples was obtained by high-throughput genotyping, and 221573 high-quality SNP sites were obtained after filtering. Combined with the phenotype information of resistance to internal white spot disease, 358 trait-associated sites were screened.

[0059] A total of 420 trait-associated SNP sites of resistance to internal white spot disease were screened by QTL mapping and GWAS, and the information of each site is shown in Table 5.

[0060] Table 5:

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Chip site design and synthesis and effectiveness verification: Based on the 420 trait-associated SNP sites of resistance to internal white spot disease, 44528 markers were screened according to the basic principle of evenly distributing SNP sites on the genome, and finally 44948 excellent SNP sites were obtained (Table 4) for subsequent probe synthesis. Thus, the P. olivaceus SNP chip for resistance to internal white spot disease can be prepared. The effectiveness of the chip was verified using 30 P. olivaceus samples. Sequencing and data analysis showed that the site detection rate of the sample data was between 98.14% and 99.26%, with an average detection rate of 98.89%, as shown in Table 6. Figure 1

[0067] Example 3:

[0068] Example 3:

[0069] ​(1) Anti-visceral white spot disease phenotype determination: Pseudomonas plecoglossicida liquid with a 120h semi-lethal dose (1x10 4 CFU / ml) was injected into the abdominal cavity of large yellow croaker (about 30g per tail), and the survival state of individuals was recorded: the median lethal time was used as the dividing line, and the individuals before the median lethal time were recorded as 0, and the rest were recorded as 1. The survival state was used as the anti-visceral white spot disease phenotype for subsequent analysis.

[0070] (2) Obtain DNA samples: Collect fin tissue samples of large yellow croaker, extract genomic DNA from the tissue by phenol-chloroform method or special reagent kit, and detect qualified DNA by agarose gel electrophoresis and Nanodrop. After adjusting the DNA concentration to the working concentration of 10 ng / μL, the SNP chip detection was carried out.

[0071] (3) Genomic chip detection: The developed SNP chip was used for genotype detection of all samples, and the detection process was operated according to the standard detection process of Genotyping By Target Sequencing (GBTS) technology of Shijiazhuang Boruitai Biotechnology Co., Ltd.

[0072] (4) Data analysis: The raw data obtained by detection was quality controlled by FastQ software, and then the sequencing data was aligned to the reference genome sequence of large yellow croaker by BWA software. The standard process of GATK software was used for SNP detection and genotyping.

[0073] The results showed that the population SNP site was between 98.10% and 99.32%, with an average detection rate of 98.98%. The genotyping data was quality controlled: individuals and markers with a deletion rate >10% and MAF <0.05 were deleted. After quality control, 42008 high-quality SNP markers were obtained, which were used for subsequent genomic selection breeding of disease resistance traits. The survival state after artificial infection was used as the large yellow croaker anti-visceral white spot disease phenotype, and the restricted maximum likelihood method (REML) was used to estimate the heritability of disease resistance traits based on the SNP information obtained by large yellow croaker anti-visceral white spot disease whole genome SNP chip genotyping. At the same time, the genomic best linear unbiased prediction (GBLUP) method was used to predict the individual genomic estimated breeding value (GEBV), and the prediction reliability of GEBV was analyzed. The heritability of large yellow croaker anti-visceral white spot disease traits was calculated to be 0.35±0.05, and the prediction reliability of GEBV was as high as 0.66, as shown in Table 6. The results prove that the large yellow croaker anti-visceral white spot disease whole genome SNP chip has high reliability for genomic selection breeding of disease resistance traits in large yellow croaker.

[0074] Table 6:

[0075]

[0076] The above merely describes preferred embodiments of the present application, and is not intended to limit the technical scope of the present application in any way. Any minor modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application shall still fall within the technical scope of the present application.

Claims

1. A dedicated SNP chip for large yellow croaker exhibiting resistance to visceral white spot disease, characterized in that, The chip includes a probe array with 44,948 SNPs, all of which are located on the genome chromosome.

2. The SNP chip for large yellow croaker's resistance to visceral white spot disease as described in claim 1, characterized in that, The number of loci corresponding to the chromosome numbers of the chromosomes are as follows: Chr01-Chr24: 2260, 1979, 1998, 1458, 2463, 1896, 2140, 2077, 1963, 1669, 1695 , 2392, 1464, 2395, 1800, 1700, 1845, 1541, 2253, 1666, 2050, 1580, 1388, 1276.

3. The SNP chip for large yellow croaker's resistance to visceral white spot disease as described in claim 1, characterized in that, The genome was obtained using PacBio SMRT and Hi-C technology from the Daqu large yellow croaker genome.

4. The SNP chip for large yellow croaker's resistance to visceral white spot disease as described in claim 1, characterized in that, Among the SNP sites, there are 420 key SNP sites for the trait of resistance to visceral white spot disease.

5. A dedicated SNP chip for large yellow croaker's resistance to visceral white spot disease as described in claim 4, characterized in that, The number of the 420 loci on each chromosome is as follows: Chr01-Chr24: 29, 29, 4, 14, 20, 12, 12, 9, 19, 26, 22, 13, 11, 26, 14, 17, 4, 11, 54, 4, 17, 17, 26, 10.

6. The application of the SNP site combinations according to claims 1-5 in the preparation of SNP chips for the whole genome of large yellow croaker.

7. The application of the SNP site combinations described in claims 1-5 in the genome selection breeding of large yellow croaker.

8. A method for whole-genome genotyping detection of large yellow croaker, characterized in that, Includes the following steps: (1) Obtain the genomic DNA of the large yellow croaker sample to be tested; (2) The genomic DNA of the large yellow croaker was detected using a whole-genome SNP chip for disease resistance traits to obtain raw data; (3) Perform data analysis on the original data to obtain the genotyping results.

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