Larimichthys crocea medium-low density SNP liquid phase chip and application thereof

Abstract

The invention provides a large yellow croaker medium-low density SNP liquid phase chip and application thereof, the chip is prepared from a probe for detecting 15,292 SNP molecular marker combinations of a large yellow croaker whole genome, and the position and base information of each SNP site on the chip are shown in a table 1. The invention further provides a preparation method and application of the chip, such as application in large yellow croaker population genetic diversity analysis, breeding population genotype identification, character correlation analysis or genome selective breeding. The chip designed by the invention comprises a series of core sites obviously related to growth, flow resistance and quality characters, and also comprises a large number of background sites, so that the detection accuracy of the designed chip is guaranteed, and the chip has the obvious advantage of low detection cost, the detection cost is reduced by more than 30%, and the detection efficiency is improved. The scientific and technological innovation of the large yellow croaker dark blue breeding industry can be further accelerated.

Description

Technical Field

[0001] The invention belongs to the field of fish genetic breeding, relates to the technical field of genome selection breeding, and specifically relates to a large yellow croaker medium- and low-density SNP liquid phase chip and an application thereof. Background Art

[0002] Large yellow croaker is known as the "national fish" and is one of the important special marine cage-cultured fish in my country, with an annual aquaculture output of over 280,000 tons. As we all know, seed industry is the foundation of industrial development. Currently, four new large yellow croaker varieties, "Minyou No. 1", "Donghai No. 1", "Yongdai No. 1" and "Fufa No. 1", have been bred one after another. However, with the development of aquaculture scale and innovation of models in recent years, the existing large yellow croaker new varieties still cannot meet the actual needs of industrial development. Therefore, it is urgent to create a series of large yellow croaker new varieties with advantageous traits for different aquaculture models to support the healthy and sustainable development of the industry.

[0003] Most of the breeding routes for the existing new varieties of yellow croaker use the method of group selection. Although varieties with excellent economic traits can be bred through traditional breeding techniques such as group and family selection, traditional breeding techniques have defects such as long cycle, low efficiency and poor predictability, which greatly restricts the pace of development of yellow croaker seed industry. With the rapid development of molecular biology and sequencing technologies, new genotyping and analysis technologies continue to emerge and are widely used in yellow croaker breeding research. Among them, SNP chip technology is a high-throughput SNP detection method developed after the traditional SNP detection method. It is currently a common and efficient and low-cost genomic breeding technology in the field of biological breeding. Chip technology is divided into solid phase chips and liquid phase chips according to the type of its carrier. As the name suggests, the sites of solid phase chips are fixed once they are designed, and sites cannot be added or deleted. They do not have good flexibility and the typing cost is high. Compared with solid phase chips, liquid phase chips based on targeted capture sequencing technology have the advantages of high cost performance and flexible customization. Liquid phase chips can be divided into high-density, medium- and low-density chips according to the number of designed SNP sites. As the name suggests, high-density liquid phase chips contain a large number of SNP sites and are universal, but they are costly and not very targeted. Medium- and low-density liquid phase chips focus on several breeding traits and have lower detection costs, so they are more practical.

[0004] At present, the existing large yellow croaker breeding chips on the market mainly include 600K, 55k solid phase chips and 55k high-density liquid phase chips. The detection cost of the above chips is generally high, so the penetration rate in the market is very low. In addition, the SNP sites enriched by the chip are closely related to the basic population used for its resequencing detection. The SNP sites enriched by a chip cannot be associated with all breeding traits. In other words, one chip cannot be suitable for all trait breeding. Promoting the steady development of deep-sea aquaculture industry has become a current strategic need of the country. However, the lack of suitable deep-sea aquaculture varieties is one of the core bottlenecks restricting the development of my country's deep-sea aquaculture industry. Large yellow croaker is one of the main marine economic fish currently expanded to deep-sea aquaculture. However, as of now, there is no new variety of large yellow croaker that is suitable for deep-sea aquaculture. Therefore, it is urgent to carry out the research and development of deep-sea aquaculture large yellow croaker breeding technology system and its variety creation. Therefore, it is urgent to develop a low-cost and efficient medium-low density SNP liquid phase chip for deep-sea aquaculture excellent trait breeding. Summary of the invention

[0005] In view of the above problems, the present invention provides a low-density SNP liquid phase chip and method for large yellow croaker. The chip contains a series of core sites significantly associated with growth, flow resistance, and quality traits, and also contains a large number of background sites, thereby ensuring the detection accuracy of the designed chip. The chip can be used for genetic diversity analysis of large yellow croaker populations, genotype identification of breeding populations, trait association analysis, and genomic selection breeding.

[0006] In order to achieve the above object, 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 a combination of 15,292 SNP molecular markers for the entire 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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[0048] All loci are evenly distributed on the chromosome ( Figure 1 ), including 1,173 core loci (including 179 growth trait-associated loci, 516 flow resistance trait-associated loci, and 478 quality trait-associated loci). The chip is used to evaluate the growth (body weight), flow resistance (top flow time) and muscle elasticity of large yellow croaker. Among them, Table 2 lists the locus information of 179 SNP loci associated with growth traits, Table 3 lists the locus information of 516 SNP loci associated with flow resistance traits, and Table 4 lists the locus information of 478 SNP loci associated with quality traits.

[0049] Table 2 SNP loci information associated with growth traits

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[0052] Table 3 Information on SNP loci associated with flow resistance

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[0057] Table 4 Information on SNP loci associated with quality traits

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[0063] In a second aspect, the present invention provides a method for preparing the above-mentioned large yellow croaker medium and low-density SNP liquid phase chip, comprising the following steps:

[0064] (1) Construction of large yellow croaker sample population

[0065] A certain number of target analysis large yellow croakers were randomly selected, and their growth, flow resistance and muscle elasticity were evaluated respectively. Then, the same number of large yellow croakers with fast growth, slow growth, strong flow resistance, weak flow resistance, high muscle elasticity and low muscle elasticity were selected.

[0066] In the specific implementation of the present invention, 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 large yellow croakers in each batch. The three traits of large yellow croakers, growth (body weight), flow resistance (top flow time) and muscle elasticity, were evaluated respectively, and then 150 individuals with fast growth, slow growth, strong flow resistance, weak flow resistance, high muscle elasticity and low muscle elasticity were selected for subsequent whole genome resequencing.

[0067] (2) Whole genome resequencing of large yellow croaker

[0068] The six constructed groups of large yellow croaker were subjected to whole genome resequencing, and the specific steps included: 1) DNA extraction; 2) DNA sample quality inspection, and resequencing library construction for DNA that passed the quality inspection; 3) after the library quality inspection passed, whole genome sequencing was performed; 4) the resequencing data was compared with the large yellow croaker reference genome sequence, and genetic marker SNP detection formed by single nucleotide variation was performed. The platform, kit or method for obtaining the large yellow croaker reference genome sequence selected in this step were all obtained from the prior art.

[0069] (3) Development of 15K liquid phase breeding chip for large yellow croaker

[0070] A) Principles for selecting candidate sites: MAF>0.1, heterozygosity rate<50%, deletion rate<10%, uniform distribution, and compliance with site evaluation principles, and probe design;

[0071] B) Site evaluation principle: Extract the upstream and downstream sequences of the SNP site, according to the probe length of 110 bp, GC content of 30-70%, and the maximum number of similar fragments of 5;

[0072] C) The sites for which probes were successfully designed were selected according to the principle of uniform distribution, and a total of 15,790 sites were selected. Subsequently, 15,292 sites were selected as the final site set based on capture stability, including 1,173 core sites (179 sites associated with growth traits, 516 sites associated with flow resistance traits, and 478 sites associated with quality traits).

[0073] The synthetic 15K site panel was tested, and after sequencing and data analysis, the sample site detection rate was between 99.48% and 99.83%, and the average detection rate was 99.71%; the consistency rate of repeated sample detection was between 99.29% and 99.75%, and the average consistency rate was 99.65%.

[0074] In addition, the SNP sites contained in this chip are highly polymorphic in various natural populations of large yellow croaker, and can be used well for genetic background analysis of large yellow croaker breeding populations.

[0075] Therefore, the third aspect of the present invention provides the application of the above-mentioned medium and low density SNP liquid phase chip for large yellow croaker. The present invention provides a set of probes for detecting the combination of SNP sites in the whole genome of large yellow croaker, which is used to capture the sites contained in the liquid phase chip provided by the present invention, and can be used for genetic diversity analysis of large yellow croaker populations, genotype identification of breeding populations, trait association analysis, and genome selection breeding, and is suitable for application in large yellow croaker seed selection, variety creation, or genotype analysis of different geographical populations.

[0076] Preferably, the application is in the genetic improvement of economic traits. Further, the economic traits are selected from any one or more combinations of body weight, flow resistance and muscle elasticity.

[0077] Functions and Effects of the Invention

[0078] (1) The number of samples used for designing the chip in the present invention is 900, and the sources are wide, which helps to improve the accuracy of trait-associated site selection and is conducive to improving the efficient breeding of large yellow croaker and the research and protection of its germplasm resources.

[0079] (2) The chip designed by the present invention contains a series of core sites significantly related to growth, flow resistance, and quality traits, and also contains a large number of background sites, thereby ensuring the detection accuracy of the designed chip.

[0080] (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, which can reduce the detection cost by more than 30%.

[0081] (4) The chip designed by the present invention focuses on the key traits required for deep-sea aquaculture of large yellow croaker, such as growth, flow resistance and quality traits, which is conducive to further accelerating the technological innovation of the large yellow croaker deep-sea breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It shows that the loci in the low-density SNP liquid phase array in large yellow croaker are evenly distributed on the chromosome;

[0083] Figure 2 The results of the microarray site detection rate are shown;

[0084] Figure 3 The chip repetition rate sample detection consistency results are shown;

[0085] Figure 4 Results of the evaluation of the prediction accuracy of the array-based genomic selection are shown;

[0086] Figure 5 It was shown that the chip can distinguish genotypes of different geographical populations. DETAILED DESCRIPTION

[0087] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0088] Example 1 Preparation of Large Yellow Croaker 15K Liquid Phase Breeding Chip

[0089] (1) Construction of large yellow croaker sample population

[0090] More than 3000 large yellow croakers were randomly selected from aquaculture groups in different sea areas of Ningde, Fujian in three batches, with about 1000 large yellow croakers in each batch. The three traits of large yellow croakers, growth (body weight), flow resistance (top flow time) and muscle elasticity, were evaluated respectively. Then, 150 individuals of each type, including those with fast growth, slow growth, strong flow resistance, weak flow resistance, high muscle elasticity and low muscle elasticity, were selected (Table 5).

[0091] Table 5 Six groups shape data

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[0097] (2) Whole genome resequencing of large yellow croaker

[0098] The whole genome of 900 large yellow croakers from the six constructed populations was resequenced. The specific steps included:

[0099] 1) Extract DNA using magnetic beads. 2) DNA sample quality inspection, DNALibrary Prep Kit for ILM was used to construct a resequencing library for DNA that passed the quality inspection. 3) After the library quality inspection was qualified, it was sequenced using the BGI-2000 / MGI-T7 sequencing platform, and the sequencing mode was PE150 mode. 4) The resequencing data was compared with the reference genome sequence of large yellow croaker (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_003845795.1 / ) using the software BWA (mem alignment mode) to detect genetic markers (SNPs) formed by single nucleotide variations.

[0100] (3) Development of 15K liquid phase breeding chip for large yellow croaker

[0101] 1) Principles for selecting candidate sites: MAF>0.1, heterozygosity rate<50%, deletion rate<10%, uniform distribution, and compliance with site evaluation principles, and the probe was successfully designed.

[0102] 2) Site evaluation principle: Extract the upstream and downstream sequences of the SNP site, according to the probe length of 110 bp, GC content of 30-70%, and the maximum number of similar fragments = 5.

[0103] 3) The sites for which probes were successfully designed were selected according to the principle of uniform distribution, and a total of 15,790 sites were selected. Subsequently, 15,292 sites were selected as the final site set based on capture stability, including 1,173 core sites (179 sites associated with growth traits, 516 sites associated with flow resistance traits, and 478 sites associated with quality traits), with an average spacing of 43.50 Kb between sites.

[0104] The selected sites were tested based on the liquid probe capture technology GenoBaits, and a 15K site panel ("Donghai Core No. 1") was designed and synthesized. After sequencing and data analysis, the sample site detection rate was between 99.48% and 99.83%, with an average detection rate of 99.71% ( Figure 2 ); the consistency of repeated sample detection was between 99.29% and 99.75%, and the average consistency was 99.65% ( Figure 3 ).

[0105] Example 2 Evaluation of the prediction accuracy of genome selection of “Donghaixin No. 1” large yellow croaker SNP liquid breeding chip

[0106] For the SNP typing data of 900 large yellow croakers for the three traits of body weight, flow resistance and muscle elasticity obtained in Example 1, markers whose genotypes covered at least 80% of the individuals were screened and markers with a minimum allele frequency (MAF) less than 0.05 were removed. At the same time, the 3δ rule was used to remove phenotypic extreme values, and finally SNP markers of 766 large yellow croakers were obtained for subsequent analysis.

[0107] 1) Estimation of trait heritability

[0108] This implementation case uses phenotypic data and genotypic data to estimate population variance and calculate the heritability of each trait. The heritability calculation formula is as follows:

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[0110] The genetic variance was estimated using the kin.blup function of the R package (rrBLUP) genetic variance) and error variance ( The error variance) was used to calculate the trait heritability according to the above formula. Table 6 shows the estimated heritability of the three traits, among which the estimated heritability of the weight trait is 0.74626, the estimated heritability of the elasticity trait is 0.79507, and the estimated heritability of the resistance trait is 0.74528.

[0111] Table 6 Estimated heritability of three traits

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[0113] 2) Genomic selection model

[0114] In this embodiment, 9 models including rrblup (implemented by mix.solve function of R language package rrBLUP), svmrbf (implemented by R language package kemlab), svmpoly (implemented by R language package kemlab), randomforest (implemented by R language package randomForest), pls (implemented by R language package pls[), gblupD (implemented by kin.blup function of R language package rrBLUP), gblupA (implemented by kin.blup function of R language package rrBLUP), BayesA (calculated by R language package BGLR), BayesB (calculated by R language package BGLR), BayesC (calculated by R language package BGLR) and BayesLasso (calculated by R language package BGLR) are used to train the genotype data and phenotypic data of the reference population, calculate the prediction accuracy through cross-validation, and then predict the phenotypic value of the prediction population.

[0115] Using the 5-fold cross-validation method, the reference population was randomly divided into 5 parts, 4 of which were used as training sets and the remaining 1 as a test set. Each sample had the opportunity to be used as a test set, and the Pearson correlation coefficient between the true value and the predicted value of the test set was calculated. The cross-validation was repeated 20 times, and the final correlation coefficient mean was used as the prediction accuracy (r). Figure 4 The accuracy calculated by all models is displayed, showing that the genomic selection prediction accuracy of the three traits is the highest under the randomforest model.

[0116] Example 3 Application of "Donghaixin No. 1" large yellow croaker SNP liquid phase breeding chip in current resistance breeding

[0117] The estimated breeding value (GEBV) of the collected and constructed 416 large yellow croaker breeding parent groups for the flow resistance trait was calculated using the random forest model in Example 2. The breeding parents were selected according to the selection pressure of 10%. Table 7 shows the GEBV values ​​of the 40 breeding parents.

[0118] Table 7 Summary of GEBV values ​​of 40 breeding parents

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[0120] Example 4 Application of "Donghaixin No. 1" large yellow croaker SNP liquid breeding chip in genotyping analysis of different geographical populations

[0121] Using the "Donghai Core No. 1" large yellow croaker SNP liquid breeding chip designed in Example 1, samples from three geographical populations of large yellow croaker (Daiqu, Minyuedong, and Naozhou) were tested. After screening markers whose genotypes covered at least 80% of the individuals and removing markers with a minimum allele frequency (MAF) of less than 0.05, genotype information of 14,716 high-quality SNP loci was obtained. Based on this data, the genetic structure of the tested large yellow croaker was clustered using the neighbor-joining method. The results are as follows: Figure 5 As shown in the figure, all the typing results of the tested large yellow croaker are consistent with the actual grouping results, and they are divided into three groups: Daiqu, Minyuedong and Naozhou. The results show that the SNP loci screened by the "Donghaixin No. 1" large yellow croaker SNP liquid breeding chip are highly representative, and the SNP loci contained in it have good polymorphism in various natural populations of large yellow croaker, which can be used to analyze the genetic background of the large yellow croaker breeding population.

[0122] The above shows and describes 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 above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached 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 molecular marker combinations for detecting the whole genome of large yellow croaker. The location and base information of each SNP site on the chip are shown in Table 1.

2. The low-density SNP liquid phase chip of large yellow croaker according to claim 1, characterized in that: The chip is used to evaluate the growth, flow resistance and muscle elasticity of large yellow croaker. Among them, the site information associated with growth traits is shown in Table 2, the site information associated with flow resistance traits is shown in Table 3, and the site information associated with quality traits is shown in Table 4.

3. The low-density SNP liquid phase chip of large yellow croaker according to claim 1, characterized in that: The growth assessment index is body weight, and the flow resistance assessment index is top flow time.

4. The method for preparing the low-density SNP liquid phase chip of large yellow croaker according to any one of claims 1 to 3, characterized in that: The steps include: (1) Construction of large yellow croaker sample population A certain number of target analysis large yellow croakers were randomly selected, and their growth, flow resistance and muscle elasticity were evaluated respectively. Then, the same number of large yellow croakers with fast growth, slow growth, strong flow resistance, weak flow resistance, high muscle elasticity and low muscle elasticity were selected. (2) Whole genome resequencing of large yellow croaker The six constructed populations of large yellow croaker were resequenced for whole genome, and the resequencing data were compared with the large yellow croaker reference genome sequence to detect genetic markers (SNPs) formed by single nucleotide variations. (3) Development of 15K liquid phase breeding chip for large yellow croaker A) Principles for selecting candidate sites: MAF>0.1, heterozygosity rate<50%, deletion rate<10%, uniform distribution, and compliance with site evaluation principles, and probe design; B) Site evaluation principle: Extract the upstream and downstream sequences of the SNP site, according to the probe length of 110 bp, GC content of 30-70%, and the maximum number of similar fragments of 5; C) The sites for which probes were successfully designed were selected according to the principle of uniform distribution, and 15,292 sites were selected as the final site set in combination with capture stability, and the average spacing between sites was set.

5. The method for preparing the low-density SNP liquid phase chip of large yellow croaker according to claim 4, characterized in that: In step (1), the number of six types of large yellow croaker individuals is not less than 100; In step (2), the specific steps of whole genome resequencing of large yellow croaker include: 1) extracting DNA; 2) quality inspection of DNA samples, and constructing a resequencing library for DNA that passes the quality inspection; 3) after the library quality inspection is qualified, whole genome sequencing is performed; 4) comparing the resequencing data with the large yellow croaker reference genome sequence, and performing genetic marker SNP detection formed by single nucleotide variation. In step (3), the average distance between sites was 43.50 Kb.

6. Use of the low-density SNP liquid phase chip of large yellow croaker according to any one of claims 1 to 3 in genetic diversity analysis of large yellow croaker populations, genotype identification of breeding populations, trait association analysis or genomic selection breeding.

7. The use according to claim 6, characterized in that: The application is application in genetic improvement of economic traits.

8. The use according to claim 7, characterized in that: The economic traits are selected from any one or more combinations of body weight, flow resistance and muscle elasticity.

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