Low-density SNP liquid phase chip in large yellow croaker and its application

Abstract

This invention provides a low-density SNP liquid-phase chip for large yellow croaker and its applications. The chip is prepared using probes to detect 15,292 SNP molecular marker combinations across the entire large yellow croaker genome. The positions and base information of each SNP site on the chip are shown in Table 1. This invention further provides a method for chip preparation and its applications, such as in the analysis of genetic diversity in large yellow croaker populations, genotyping of breeding populations, trait association analysis, or genomic selection breeding. The chip designed in this invention contains a series of core loci significantly associated with growth, flow resistance, and quality traits, while also including a large number of background loci, ensuring the accuracy of the chip's detection. It has a significant advantage in low detection cost, reducing detection costs by more than 30%, which is conducive to further accelerating technological innovation in the deep-blue seed industry of large yellow croaker.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic breeding, specifically the field of genome selection breeding technology, and relates to a low-density SNP liquid phase chip for large yellow croaker and its application. Background Technology

[0002] As we all know, the seed industry is the foundation of industrial development. Currently, four new varieties of large yellow croaker, namely "Minyou No. 1", "Donghai No. 1", "Yongdai No. 1" and "Fufa No. 1", have been successively cultivated. However, with the development of aquaculture scale and the innovation of models in recent years, the existing new varieties of large yellow croaker still cannot meet the actual needs of industrial development. Therefore, it is urgent to create a series of new varieties of large yellow croaker with advantageous traits for different aquaculture models to support the healthy and sustainable development of the industry.

[0003] Currently, most breeding routes for new large yellow croaker varieties employ population selection methods. While traditional breeding techniques such as population and family selection can produce varieties with excellent economic traits, these methods suffer from drawbacks such as long cycles, low efficiency, and poor predictability, significantly limiting the development of the large yellow croaker breeding industry. With the rapid development of molecular biology and sequencing technologies, novel genotyping and analysis techniques are constantly emerging and widely applied in large yellow croaker breeding research. Among these, SNP microarray technology, a high-throughput SNP detection method developed after traditional SNP detection methods, is currently a commonly used, efficient, and low-cost genomic breeding technology in the field of bio-breeding. Microarray technology is divided into solid-phase microarrays and liquid-phase microarrays based on the type of carrier. Solid-phase microarrays, as the name suggests, have fixed loci that cannot be added or deleted, lacking flexibility and having high genotyping costs. Compared to solid-phase microarrays, liquid-phase microarrays, based on targeted capture sequencing technology, offer advantages such as high cost-effectiveness and flexible customization. Liquid phase chips can be divided into high-density and medium-low density chips based on the number of SNP sites designed. As the name suggests, high-density liquid phase chips contain more SNP sites and are more versatile, but they are more expensive and less targeted. Medium-low density liquid phase chips focus on a few selective traits and have lower detection costs, so they are more practical.

[0004] Currently available large yellow croaker breeding chips mainly include 600K and 55K solid-phase chips and 55K high-density liquid-phase chips. The detection costs of these chips are generally high, resulting in low market penetration. Furthermore, the SNP sites enriched by the chip are closely related to the base population used for resequencing detection; a single chip cannot be associated with all selected traits, meaning it cannot be suitable for breeding all traits. Large yellow croaker is one of the main marine economic fish species currently being promoted for deep-sea aquaculture, but to date, no new large yellow croaker varieties adapted to deep-sea aquaculture have been developed. Therefore, there is an urgent need to develop a breeding technology system for large yellow croaker in deep-sea aquaculture and to create new varieties. Thus, there is an urgent need to develop a low-cost, high-efficiency, low-density SNP liquid-phase chip for large yellow croaker used for selecting superior traits in deep-sea aquaculture. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a low-density SNP liquid phase chip and method for large yellow croaker. This chip contains a series of core loci significantly associated with growth, flow resistance, and quality traits, as well as a large number of background loci, ensuring the detection accuracy of the designed chip. It can be used for genetic diversity analysis of large yellow croaker populations, genotype identification of breeding populations, trait association analysis, and genome selection breeding.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] In a first aspect, the present invention provides a low-density SNP liquid phase chip for large yellow croaker, which is prepared by probes for detecting 15,292 SNP molecular marker combinations in the whole genome of large yellow croaker. The position and base information of each SNP site on the chip are shown in Table 1.

[0008] Table 1 Summary of SNP site locations and base information

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[0101] All loci are evenly distributed on the chromosome. Figure 1 The chip contains 1,173 core loci (179 associated with growth traits, 516 associated with current resistance traits, and 478 associated with quality traits). This chip is used to evaluate three traits in large yellow croaker: growth (body weight), current resistance (time spent in the current), and muscle elasticity. Table 2 lists the locus information associated with the 179 SNPs and growth traits, Table 3 lists the locus information associated with the 516 SNPs and current resistance traits, and Table 4 lists the locus information associated with the 478 SNPs and quality traits.

[0102] Table 2. SNP loci associated with growth traits

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[0106] Table 3. SNP information associated with flow resistance traits

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[0112] Table 4. SNP loci associated with quality traits

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[0117] In a second aspect, the present invention provides a method for preparing a low-density SNP liquid-phase chip in large yellow croaker as described above, comprising:

[0118] The following steps are required:

[0119] (1) Construction of large yellow croaker sample population

[0120] A certain number of target large yellow croakers were randomly selected for analysis. The growth, resistance to current and muscle elasticity were evaluated. Then, the same number of large yellow croakers of six categories were selected: fast growth, slow growth, strong resistance to current, weak resistance to current, high muscle elasticity and low muscle elasticity.

[0121] In the specific implementation section of this invention, over 3,000 large yellow croakers, approximately 1,000 per batch, were randomly selected from aquaculture populations in different sea areas of Ningde, Fujian Province, in three batches. Three traits were evaluated: growth (body weight), current resistance (time spent in the current), and muscle elasticity. Then, 150 individuals each of the following traits were selected for subsequent whole-genome resequencing: fast growth, slow growth, strong current resistance, weak current resistance, high muscle elasticity, and low muscle elasticity.

[0122] (2) Whole genome resequencing of large yellow croaker

[0123] Whole-genome resequencing was performed on the six constructed populations of large yellow croaker. The specific steps included: 1) DNA extraction; 2) DNA sample quality control, and construction of resequencing libraries from the qualified DNA samples; 3) Whole-genome sequencing after the library quality control was passed; 4) Alignment of the resequencing data with the large yellow croaker reference genome sequence and detection of SNPs (single nucleotide variants). The platform, reagent kits, and methods for obtaining the large yellow croaker reference genome sequence used in this step were all obtained from existing technologies.

[0124] (3) Development of 15K liquid phase breeding chip for large yellow croaker

[0125] A) Selection criteria for candidate sites: MAF > 0.1, heterozygosity < 50%, deletion rate < 10%, uniform distribution, and compliance with site evaluation principles; probe design.

[0126] B) Site evaluation principles: Extract upstream and downstream sequences of SNP sites, according to probe length 110bp, GC content 30-70%, and maximum number of similar fragments 5;

[0127] C) Sites for successfully designed probes were selected according to the principle of uniform distribution, and a total of 15,790 sites were selected. Then, based on the capture stability, 15,292 sites were selected as the final site set, including 1,173 core sites (179 growth trait-related sites, 516 flow resistance trait-related sites, and 478 quality trait-related sites).

[0128] The synthesized 15K site panel was tested. After sequencing and data analysis, the detection rate of the sample site was between 99.48% and 99.83%, with an average detection rate of 99.71%. The consistency rate of the duplicate samples was between 99.29% and 99.75%, with an average consistency rate of 99.65%.

[0129] Furthermore, the SNP loci contained in this chip exhibit good polymorphism in various natural populations of large yellow croaker, making it well-suited for genetic background analysis of large yellow croaker breeding populations.

[0130] Therefore, in a third aspect, the present invention provides the application of the low-density SNP liquid-phase chip in large yellow croaker as described above.

[0131] The invention provides a set of probes for detecting SNP locus combinations in the whole genome of large yellow croaker, which are used to capture the loci contained in the liquid phase chip provided by the invention. It can be used for genetic diversity analysis of large yellow croaker populations, genotype identification of breeding populations, trait association analysis, and genome selection breeding. It is suitable for application in the breeding of large yellow croaker seedlings, variety creation, or genotype analysis of different geographical populations.

[0132] Preferably, the application is in the genetic improvement of economic traits. Further, the economic trait is selected from body weight,

[0133] Any one or more combinations of resistance to flow and muscle elasticity.

[0134] The role and effect of invention

[0135] (1) The number of samples used in the design of the chip in this invention is 900, and the sources are wide, which helps to improve the accuracy of the selection of trait-related sites and is conducive to improving the efficiency of breeding of large yellow croaker and the research and protection of its germplasm resources.

[0136] (2) The chip designed in this invention contains a series of core sites that are significantly related to growth, flow resistance and quality traits, as well as a large number of background sites, which ensures the detection accuracy of the designed chip.

[0137] (3) Compared with existing solid phase chips, it has the characteristics of simple operation, low cost and high flexibility. Compared with existing high-density liquid phase chips, it has the obvious advantage of low detection cost, reducing detection cost by more than 30%.

[0138] (4) The chip designed in this invention focuses on the key traits required for deep-sea aquaculture of large yellow croaker, such as growth, resistance to current and quality traits, which is conducive to further accelerating the technological innovation of deep-sea seed industry of large yellow croaker. Attached Figure Description

[0139] Figure 1 The low-density SNP sites in the liquid phase chip of large yellow croaker were shown to be uniformly distributed on the chromosome;

[0140] Figure 2 The chip site detection rate results are displayed;

[0141] Figure 3 The results show the consistency of chip repeatability sample detection.

[0142] Figure 4 The results of the accuracy assessment of microarray genome selection prediction are shown;

[0143] Figure 5 The chip demonstrated its ability to distinguish genotypes from different geographical groups. Detailed Implementation

[0144] The present invention will now be described in detail with reference to embodiments and accompanying drawings. However, the following embodiments should not be construed as limiting the scope of the present invention.

[0145] Example 1: Preparation of 15K liquid phase breeding chip for large yellow croaker

[0146] (1) Construction of the large yellow croaker sample population

[0147] More than 3,000 large yellow croakers were randomly selected from aquaculture groups in different sea areas of Ningde, Fujian Province in three batches, with about 1,000 fish in each batch. The growth (body weight), current resistance (time against the current), and muscle elasticity were evaluated. Then, 150 individuals each of the following categories were selected: fast growth, slow growth, strong current resistance, weak current resistance, high muscle elasticity, and low muscle elasticity (Table 5).

[0148] Table 5. Shape data of six groups

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[0155] (2) Whole genome resequencing of large yellow croaker

[0156] Whole-genome resequencing was performed on 900 large yellow croakers from the six established populations. The specific steps included:

[0157] 1) Extract DNA using magnetic beads. 2) Perform quality control on the DNA samples. Construct a resequencing library from the qualified DNA using the GenoBaits® DNA Library Prep Kit for ILM. 3) After the library passes quality control, sequence the DNA using the BGI-2000 / MGI-T7 sequencing platform in PE150 mode. 4) Use BWA (mem alignment) software to align the resequencing data with the large yellow croaker reference genome sequence (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_003845795.1 / ) to detect single nucleotide variants (SNPs).

[0158] (3) Development of 15K liquid phase breeding chip for large yellow croaker

[0159] 1) Selection criteria for candidate sites: MAF > 0.1, heterozygosity < 50%, deletion rate < 10%, uniform distribution, meeting the site evaluation criteria, and probes were successfully designed.

[0160] 2) Site evaluation principles: Extract upstream and downstream sequences of SNP sites, and evaluate them according to the following criteria: probe length 110bp, GC content 30-70%, and maximum number of similar fragments = 5.

[0161] 3) Sites for successfully designed probes were selected according to the principle of uniform distribution, and a total of 15,790 sites were selected. Then, based on the capture stability, 15,292 sites were selected as the final site set, including 1,173 core sites (179 growth trait-related sites, 516 flow resistance trait-related sites, and 478 quality trait-related sites), with an average spacing of 43.50 kb between sites.

[0162] The selected loci were used to design and synthesize a 15K locus panel (“Donghai Core 1”) based on the GenoBaits liquid-phase probe capture technology for testing. Sequencing and data analysis showed that the locus detection rate ranged from 99.48% to 99.83%, with an average detection rate of 99.71%. Figure 2 The consistency rate of duplicate samples was between 99.29% and 99.75%, with an average consistency rate of 99.65%. Figure 3 ).

[0163] Example 2: Accuracy Assessment of Genomic Selection Prediction Using the "Donghai Core 1" Large Yellow Croaker SNP Liquid Phase Breeding Chip

[0164] For the SNP genotyping data of 900 large yellow croakers with three traits of body weight, flow resistance and muscle elasticity obtained in Example 1, markers covering at least 80% of individuals were screened and markers with a minimum allele frequency (MAF) of less than 0.05 were removed. At the same time, the 3δ rule was used to remove phenotypic extreme values, and finally the SNP markers of 766 large yellow croakers were obtained for subsequent analysis.

[0165] 1) Estimation of heritability of traits

[0166] This implementation case uses phenotypic and genotypic data to estimate population variance and calculate the heritability of each trait. The formula for calculating heritability is as follows:

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[0168] Use the kin.blup function of the R package (rrBLUP) to estimate genetic variance. (genetic variance) and error variance (error variance), and the heritability of the traits was calculated according to the above formula. Table 6 shows the estimated heritability of the three traits, with the estimated heritability of weight being 0.74626, elasticity being 0.79507, and flow resistance being 0.74528.

[0169] Table 6. Estimated heritability of the three traits

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[0171] 2) Genomic selection model

[0172] This embodiment uses nine models—rrblup (implemented using the mix.solve function of the rrBLUP R package), svmrbf (implemented using the kemlab R package), svmpoly (implemented using the kemlab R package), randomforest (implemented using the randomForest R package), pls (implemented using the pls[ R package), gblupD (implemented using the kin.blup function of the rrBLUP R package), gblupA (implemented using the kin.blup function of the rrBLUP R package), BayesA (calculated using the BGLR R package), BayesB (calculated using the BGLR R package), BayesC (calculated using the BGLR R package), and BayesLasso (calculated using the BGLR R package)—to train the genotype and phenotypic data of the reference population. The prediction accuracy is calculated through cross-validation, and then phenotypic values ​​are predicted for the target population.

[0173] A 5-fold cross-validation method was used, in which the reference population was randomly divided into 5 parts, with 4 parts serving as the training set and the remaining part as the test set. Each sample had a chance to be included in the test set, and the Pearson correlation coefficient between the actual and predicted values ​​in the test set was calculated. The cross-validation was repeated 20 times, and the mean of the final correlation coefficients was used as the prediction accuracy (r). Figure 4 The accuracy calculated by all models is shown, and the genomic selection prediction accuracy for all three traits is the highest under the randomforest model.

[0174] Example 3: Application of the "Donghai Core No. 1" large yellow croaker SNP liquid phase breeding chip in anti-current breeding

[0175] The estimated breeding value (GEBV) for the current resistance trait of the 416 large yellow croaker breeding parent population was calculated using the randomforest model in Example 2. Breeding parents were selected with a selection pressure of 10%, and Table 7 shows the GEBV values ​​of 40 breeding parents.

[0176] Table 7 Summary of GEBV values ​​of 40 breeding parents

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[0179] Example 4: Application of the "Donghai Core No. 1" large yellow croaker SNP liquid phase breeding chip in genotyping of different geographical populations

[0180] Using the "Donghai Core 1" large yellow croaker SNP liquid-phase breeding chip designed in Example 1, samples from three geographical populations of large yellow croaker (Daiqu, Minyuedong, and Naozhou) were tested. After screening for markers whose genotypes covered at least 80% of individuals and removing markers with a minimum allele frequency (MAF) of less than 0.05, genotypic information for 14,716 high-quality SNP loci was obtained. Based on this data, the genetic structure of the tested large yellow croaker was clustered using neighbor-joining methods, and the results are as follows. Figure 5 As shown, all the genotyping results of the large yellow croaker tested were consistent with the actual grouping results, dividing them into three groups: the Daiqu tribe, the Min-Yue Dong tribe, and the Naozhou tribe. This result indicates that the SNP loci screened by the "Donghai Core 1" large yellow croaker SNP liquid-phase breeding chip are highly representative, and the included SNP loci exhibit good polymorphism in all natural populations of large yellow croaker, making it a good tool for analyzing the genetic background of large yellow croaker breeding populations.

[0181] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A low-density SNP liquid phase chip for large yellow croaker, characterized in that, The probes were prepared from 15,292 SNP combinations that detect the whole genome of large yellow croaker. The positions and base information of each SNP site on the chip are shown in Table 1 of the instruction manual. The reference genome is GCA_003845795.

1.

2. The low-density SNP liquid phase chip for large yellow croaker according to claim 1, characterized in that, The chip is used to evaluate three traits of large yellow croaker: growth, resistance to currents, and muscle elasticity. The locus information associated with growth traits is shown in Table 2 of the instruction manual, the locus information associated with flow resistance traits is shown in Table 3 of the instruction manual, and the locus information associated with muscle elasticity traits is shown in Table 4 of the instruction manual. The growth status is assessed by body weight, and the resistance to flow is assessed by time spent in the flow.

3. The application of the low-density SNP liquid phase chip in large yellow croaker according to any one of claims 1 to 2 in the analysis of genetic diversity in large yellow croaker populations, genotyping of breeding populations, or genomic selection breeding, characterized in that, The breeding traits refer to three characteristics of the large yellow croaker: growth, resistance to currents, and muscle elasticity. The growth status is assessed by body weight, and the resistance to flow is assessed by time spent in the flow.

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