Commercial pig breed 60K liquid phase chip based on targeted capture sequencing and preparation method thereof

Abstract

The invention discloses design, development and application of a commercial pig breed 60K liquid phase chip based on targeted capture sequencing. The probe target spot of the chip is screened based on longbai, large-bai and duroc whole genome re-sequencing data. The 60K liquid chip prepared by the invention can be used for commercial pig breed genetic typing, the obtained result can be used for genetic diversity evaluation, genome selection, whole genome association analysis, pig genetic improvement and other aspects, and the problems of high cost, inflexibility in probe addition and deletion, low detection speed and incapability of being used in a pig farm in a large scale in the prior art are solved. Meanwhile, the 60K liquid phase chip prepared by the invention contains SNP sites which are remarkably associated with pig economic traits and are verified by large-scale groups, and can be directly used for breeding and genetic improvement of pig target traits.

Description

Technical Field

[0001] The present invention relates to the fields of animal gene molecular breeding and genotyping technology, and specifically to a genotyping by targeted sequencing (GBTS) technology, which is applied to the design and development of a 60K liquid chip for pigs. Background Art

[0002] Single nucleotide polymorphism (SNP), as the latest generation of molecular markers, has been widely applied in the fields of agricultural breeding, medical diagnosis, etc. due to its advantages such as large quantity, wide distribution, rich polymorphism, and easy detection. With the rapid development of molecular biology and related disciplines, the genome sequencing of more and more species has been completed one after another, and high-throughput SNP detection technologies have emerged as the times require to meet the growing demand for SNP detection at the genomic level.

[0003] High-throughput SNP detection technologies mainly adopt two strategies: chips and sequencing. Most of the current mainstream SNP chips on the market are developed based on solid-phase chip technology, with relatively high prices, which limits their wide application in breeding enterprises. For example, the current mainstream 50K chips based on the Illumina platform (GGP50K of Neogen Corporation in the United States or Zhongxin No. 1 (KPSPBC50K)) have relatively high detection prices. If genotype detection is carried out on a large scale, the breeding cost is too high for breeding enterprises to bear. In addition, solid-phase chips have poor flexibility. Once designed, it is difficult to add or delete sites. Moreover, the current solid-phase chips have SNP site designs mostly based on different breeds, resulting in site redundancy or deficiency for the mainstream pig breeds Landrace, Yorkshire, and Duroc. Furthermore, the detection speed of solid-phase chips cannot meet the breeding needs. Most enterprises generally castrate young boars at 10-14 days old after piglets are born. Therefore, it is required to select the remaining individuals according to the genomic breeding value before 14 days old, and then conduct performance measurement in the later stage, and castrate the inferior boars. However, the current chip detection speed is generally slow, making it difficult to keep up with the breeding rhythm of enterprises, which technically affects the application of genomic selection. Therefore, it is necessary to develop chips with low prices, flexible customization, and relatively fast detection speeds.

[0004] With the development of sequencing technology, genotype detection (GBTS) technology based on targeted capture sequencing has brought hope for low-cost chip customization. Targeted capture sequencing is a technology for deep sequencing of specific genomic regions. Different from whole-genome sequencing, targeted capture sequencing only sequences specific loci of interest, thus significantly reducing the sequencing cost and improving the detection sensitivity of target loci at the same time. This technology designs capture probes to bind to DNA fragments in the target region, and then reads the sequence information of these fragments through a high-throughput sequencing platform. Compared with traditional SNP chips, targeted capture sequencing has higher flexibility. Users can customize the probe set according to specific research needs to achieve precise detection and analysis of specific loci. Therefore, this technology has the advantages of flexible operation, accurate and reliable, low cost, wide application range, etc. In the application of pig breeding, pig 50K liquid chips, Hainan local pig 55K liquid chips, Hunan local pig 60K chips, etc. have been developed using GBTS technology. However, existing chips still cannot meet the SNP detection needs of the mainstream pig breeds of Landrace, Large White, and Duroc. First, the genomic differences between local pig breeds and commercial pig breeds are relatively large, and local pig breed chips are not applicable to commercial pig breeds. Although China Agricultural University has designed and developed a 50K chip for commercial pig breeds, the filling effect of this chip with existing commonly used commercial chips (such as GGP50K or KPSPBC50K, etc.) is not very ideal, which will inevitably cause waste of data already sequenced with solid-phase chips. Second, the number of functional loci included in existing chips is insufficient, and there are few functional loci verified in large-scale populations. Based on the above situation, the present invention has developed a 60K liquid chip for commercial pig breeds with completely independent intellectual property rights in China. In addition to retaining the advantages of high detection accuracy and low price of GBTS, it also has the following advantages: high filling accuracy with existing chips and can be compatible with data of other chips; 2951 functional loci verified based on large-scale populations are added, which can be directly used for marker-assisted selection; flexible customization, and functional loci can be added at any time on this basis; a small number of samples can also be detected; fast detection speed and can be used for early genomic selection. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of a 60K liquid chip for commercial pig breeds designed and developed based on targeted capture sequencing technology. Based on the present invention, problems such as high cost, large sample size required, inflexible customization, lack of functional loci, and slow detection speed for genotyping of the mainstream pig breeds of Landrace, Large White, and Duroc can be solved.

[0006] In the first aspect of the present invention, a preparation method of a 60K liquid chip for commercial pig breeds based on targeted capture sequencing is provided, which mainly includes the following steps: S1: SNP site screening: Based on the whole genome resequencing data of the target population, combined with the existing commonly used pig chip sites and functional sites of known economic traits of pigs, sites are screened on the basis of ensuring uniform distribution on each chromosome.

[0007] S2: Probe design: Design and synthesize liquid phase probes based on the sites screened in step S1. The synthesized probes must meet the following conditions: probe length is 110bp; the GC content of general sites is controlled between 30% and 70% to ensure stability and hybridization efficiency; the GC content of functional sites is accepted to be 20% to 80%; the number of homologous regions cannot be more than 5, and the selected regions should avoid simple sequence repeats and blank areas as much as possible to reduce nonspecific binding; for the screened SNP sites, design two nucleotide sequences with 60%-70% overlap covering the SNP site to ensure the capture of the site.

[0008] S3: Probe synthesis: Synthesize two DNA nucleotide sequences with a length of 110 bp and a biotin group modified at the 5' end according to the nucleotide sequence designed in the above requirements. Mix the synthesized pig chip probes with equal molar mass and dilute them with EDTA and Tris-HCl buffer to a final concentration of 3 pmol / mL to prepare a chip probe mixture for subsequent experiments.

[0009] S4: Determine the final probe: Use the test sample to perform probe detection, delete the sites with low capture efficiency based on the test sample data, determine and retain the final valid and available sites, which is the pig 60K liquid phase chip.

[0010] Furthermore, the specific steps for SNP site screening of S1 are as follows: ① Collect samples of the target population for whole genome resequencing to obtain the genomic variation information of the target population; ② Select common sites (with MAF>0.1 in the target population) from the existing commonly used medium-density chips for pigs as the skeleton of the designed sites, and fill the background sites in the areas outside the common sites; ③ Add 1,000 breed-specific SNPs to each breed; ④ Add functional sites that are closely related to pig production traits; ⑤ Add the remaining sites to about 60K, ensuring uniform distribution, avoiding linkage sites, and avoiding complex genomic regions.

[0011] Further, the specific steps for S4 to determine the final probes are as follows: ① Extract the DNA of the pig sample to be tested and construct a library. Specifically, extract the DNA of the pig to be tested and use the GenoBaits DNA-seq Library Prep Ki kit (MolBreedimg Biotechnology Co, Shijiazhuang, Hebei, China) to construct a high-throughput sequencing library for pig DNA; ② Measure the quality of the constructed library. Specifically, perform quantitative analysis using a Qubit 2.0 Fluorometer, and at the same time detect the quality of the library fragments using agarose gel electrophoresis to ensure that the DNA > 500 bp and the DNA fragment range is 300 - 500 bp; ③ Capture and sequence. Specifically, mix the probes with the hybridization buffer and place them in a test tube for PCR reaction. The captured target fragments are then amplified using library amplification primers and high-fidelity DNA polymerase, followed by two rounds of purification. Finally, use Qubit and qPCR technologies to measure the concentration of the purified library, and perform sequencing using PE150 on the MGISEQ-2000 sequencing platform; ④ Determine the final probes and locus set. Specifically, further evaluate the SNP loci according to the capture situation of the test samples. The evaluation indicators include a locus deletion rate ≤ 10%, a heterozygous mutation rate ≤ 30%, and a MAF ≥ 0.01. Delete the loci with low capture efficiency among the selected loci, and determine and retain the final effective and available loci.

[0012] In the second aspect of the present invention, there is provided an application of the above-mentioned targeted capture sequencing chip in pig genome typing, and the specific steps are as follows: S1. Construct a high-throughput sequencing library for pig DNA: Extract the genomic DNA of the pig to be tested and fragment it, aiming to generate DNA fragments suitable for high-throughput sequencing; complete the construction of a high-quality sequencing library by adding sequencing adapters and performing end repair, and detect the quality of the library to ensure that it meets the sequencing requirements; S2. Capture the target genomic fragments: Combine the commercially available 60K liquid chip for pig breeds prepared as described in claim 3 with the pig DNA sequencing library constructed in step S1, and use the probes in the chip to hybridize and bind to the target genomic fragments; use a magnetic bead separation system to enable the DNA fragments containing the target SNP loci in the targeted capture library; after the washing step, remove the non-target DNA fragments and only retain the DNA in the target region; S3. Amplify, purify and perform high-throughput sequencing: The target DNA fragments captured in step S2 are amplified by PCR, the amplified products are purified, and sequencing is performed through a high-throughput sequencing platform; S4. Genotyping results: After processing and aligning the data generated by sequencing, finally obtain the accurate SNP genotyping results of the pig genome.

[0013] Further, the specific method for constructing the porcine DNA high-throughput sequencing library in S1 is as follows: Extract the genomic DNA of the porcine sample to be tested and physically fragment it to generate DNA fragments of uniform length. Subsequently, through the steps of end repair and A-tail addition, the purpose of adding appropriate sequencing adapters to the fragments is achieved, and then a high-throughput sequencing library is constructed. The quality of the library is evaluated by precision detection equipment to ensure the success of subsequent capture and sequencing.

[0014] Further, the capture method in S2 is as follows: Combine the constructed porcine DNA high-throughput sequencing library with a pre-designed porcine 60K liquid chip. The probes in the chip bind to specific genomic fragments through hybridization, and then the streptavidin-biotin system is used to capture the target fragments onto magnetic beads. This operation ensures that the captured genomic fragments contain only the sequences related to the target SNP sites through precise hybridization and enrichment. Fragments in non-target regions will be removed through the washing step.

[0015] Further, in S3, the captured target fragments are amplified by PCR to increase the copy number of the target DNA. The amplified products are purified, and then the sequence information of the target fragments is read through a high-throughput sequencing platform. After preliminary quality control of the sequencing data, professional bioinformatics software is used for analysis, and the obtained reads are aligned to the porcine reference genome for genotyping and variant detection.

[0016] In the third aspect of the present invention, there is provided the use of the chip in the evaluation of porcine genetic diversity, breed identification, genomic selection, genome-wide association analysis, and porcine genetic improvement.

[0017] In the fourth aspect of the present invention, it is provided that the chip can directly screen 2,951 loci that are significantly associated with the main economic traits of Landrace, Large White, and Duroc pigs, and these loci can be directly used for the breeding and genetic improvement of porcine target traits.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a porcine high-throughput SNP60K probe for genotyping based on targeted capture sequencing. When designing the probe, the distribution of the captured SNP sites in the whole genome is considered, and the sites selected also take into account the overlapping sites with existing chips (GBTS50K, GGP50K, KPSPBC50K), having better compatibility and practicability.

[0019] 2. The probe length designed in the present invention is 110 bp, which can ensure that the probe has sufficient length to specifically hybridize with the target sequence while avoiding non-specific binding caused by excessive length; the GC content of the probe functional site is between 20% and 80% (including the boundaries), and the other sites are between 30% and 70% (including the boundaries). This reasonable ratio can ensure the stability and hybridization rate of the probe; the number of homology regions is ≤5, and the number of homologous individuals does not exceed five to ensure the specificity of the probe and reduce the possibility of non-specific hybridization.

[0020] 3. The present invention is developed and designed based on the polymorphic sites of three major commercial pig breeds in Shandong Province, namely Changbai, Large White and Duroc, and takes into account the uniform distribution of the sites and breed-specific sites. It can be directly used for genome-wide association analysis and genome selection of major commercial pig breeds, and is more suitable for genetic improvement and resource assessment of commercial pig breeds in Shandong Province, with stronger pertinence. It provides good support for the cultivation of new white pig strains and matching strains with unique characteristics in Shandong Province, and has pioneering significance in the genetic improvement and joint breeding process of pigs in Shandong Province.

[0021] 4. The present invention designs probes based on targeted capture sequencing technology. While obtaining the genotype information of the target site, it can also perform SNP typing on the area near the target site, thereby obtaining more marker information.

[0022] 5. Compared with solid-phase chips, targeted capture sequencing technology can add or remove probes more flexibly, reducing the cost and time of probe redesign and synthesis.

[0023] 6. The chip loci of the present invention include 2951 functional loci based on large-scale population verification. Therefore, the chip can not only be used for pig genotyping, whole genome association analysis, genome selection, genetic diversity evaluation, etc., but also can directly screen out marker loci related to economic traits and can be directly used for marker-assisted selection, laying a good foundation for genetic improvement of pigs and improving the competitiveness of improved breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Filling accuracy assessment for low-depth sequencing data. a: genotype consistency; b: accuracy.

[0025] Figure 2 The distribution of the loci of the pig 60K liquid phase chip provided by the present invention. a: SNP density map; b: SNP number of different chromosomes; c: SNP spacing of different chromosomes; d: SNP MAF distribution of different breeds.

[0026] Figure 3 This is the distribution of the pig 60K liquid phase chip SNP markers in the gene structure provided by the present invention.

[0027] Figure 4 Quality assessment of genotyping of the 60K liquid-phase chip for pigs provided by the present invention. a: Detection rate; b: Stability.

[0028] Figure 5 PCA graph of the 60K liquid-phase chip for pigs provided by the present invention.

[0029] Figure 6 Overlap of autosomal loci between the 60K liquid-phase chip for pigs provided by the present invention and three existing commonly used chips (GBTS50K, GGP50K, and KPSPBC50K). Note: SDAU-PIG-GBTS60K: The 60K liquid-phase chip for pigs provided by the present invention.

[0030] Figure 7 Filling effect between the 60K liquid-phase chip for pigs provided by the present invention and three existing commonly used chips. a-c: Filling the other three chips into the 60K liquid-phase chip for pigs provided by the present invention; e-f: Filling the 60K liquid-phase chip for pigs provided by the present invention into the other three chips.

[0031] Figure 8 Accuracy of filling the four chips into sequencing data. Note: SDAU-PIG-GBTS60K: The 60K liquid-phase chip for pigs provided by the present invention.

[0032] Figure 9 Evaluation of the genomic selection effect of the 60K liquid-phase chip for pigs provided by the present invention. Detailed implementation mode

[0033] Next, the technical solutions in the embodiments of the present invention will be described in combination with the implementation examples of the present invention. Obviously, the described examples are only part of the examples of the present invention, rather than all the examples.

[0034] Example 1: Design of a 60K liquid-phase chip for commercial pig breeds based on targeted capture sequencing The molecular markers described in the present invention are based on the re-sequencing data of a total of 5928 samples of three breeds, Landrace, Yorkshire, and Duroc, in Shandong Province. On this basis, loci are screened and probes are designed and synthesized. The specific steps are as follows: S1. Whole-genome re-sequencing of the target pig breed: The information of target SNP loci is derived from the re-sequencing data of 5928 commercial pig breeds in Shandong Province, specifically 1306 Landrace, 3145 Yorkshire, and 1477 Duroc. After collecting blood and extracting DNA, whole-genome low-depth sequencing is performed based on the BGI-sequencing T7 sequencing platform, and the average sequencing depth is 1.5X.

[0035] S2. Sequence preprocessing and SNP detection: The raw data obtained from sequencing was preprocessed to filter out invalid and low-quality data, resulting in clean reads. The clean reads were aligned to the pig reference genome (Sscrofa11.1) using the BWA v0.7.17 tool. The resulting BAM file was sorted using the SAMtools v1.11 tool, and PCR duplicates were removed using the Picard tool. SNP calling was performed using the BaseVar v0.8.0 tool, followed by genotype imputation using the STITCH v1.6.10 tool. The results of STITCH were imputed a second time using Beagle v5.4. 22,085,320, 23,276,520, and 19,363,918 SNPs were identified in Landrace, Large White, and Duroc pigs, respectively. The imputation accuracy is as Figure 1 shown, with both genotype consistency and accuracy being relatively high, which can be used for subsequent SNP locus screening.

[0036] S3. SNP locus screening: ① Based on the identified SNP loci above, 12,309 common loci were selected from the existing available chips (CAU50K, GGP50K, and KPSPBC50K) as the backbone for designing loci, and background loci were filled in the regions outside the common loci. ② Appropriate allele frequency: To ensure the effectiveness of chip detection, the added loci on the chip are required to have MAF > 0.35. ③ To ensure breed compatibility, 1000 breed-specific SNPs were added for each breed. ④ Functional loci were added, and the functional loci were 2951 SNPs significantly associated with pig economic traits identified by the project team in the early stage. Specifically, first, SNPs significantly associated with multiple traits were selected, requiring MAF > 0.1; then, the top five SNPs with the highest significance level for each trait were selected and MAF > 0.05; finally, SNPs significantly associated with two breeds and MAF > 0.2 were selected. ⑤ Based on the uniformity of locus distribution, the remaining loci were added to ensure the effectiveness of subsequent association analysis. ⑥ Avoid linked loci: The linked blocks were calculated, and only the first few longest linked blocks were screened within an interval, and only one locus was screened in one block to avoid probe waste caused by linked loci. ⑦ Avoid genomic complex regions: Loci in simple repeat sequence regions, loci with flanking sequence homology greater than 5, and loci with a GC content of 30 - 70% were filtered out to avoid probe capture failure for complex region loci.

[0037] S4. Design and synthesis of liquid-phase probes. The design principles are as follows: ① The probe length is 110 bp; ② The GC content of general sites is controlled between 30% and 70% to ensure stability and hybridization efficiency; the GC content of functional sites is allowed to be 20 - 80%. ③ The number of homologous regions should not exceed 5; ④ Design two nucleotide sequences with 60% - 70% overlapping coverage of the selected SNP sites. Synthesize two DNA nucleotide sequences with a length of 110 bp and a biotin group modification at the 5' end according to the above requirements, which are the probes for the porcine 60K chip. Mix the synthesized porcine chip probes with equal molar mass and dilute them with EDTA and Tris-HCl buffer to a final concentration of 3 pmol / mL to prepare a chip probe mixture.

[0038] S5. Genotyping using a liquid-phase chip. This step is divided into four processes: extracting DNA from the porcine sample to be tested for library construction, measuring the quality of the constructed library, capturing and sequencing, and determining the final probe.

[0039] ① The specific operation for extracting DNA from the porcine sample to be tested for library construction is as follows: Extract the DNA of the porcine to be tested and use the GenoBaits DNA-seq Library Prep Ki kit (MolBreedimg Biotechnology Co, Shijiazhuang, Hebei, China) to construct a high-throughput porcine DNA sequencing library.

[0040] ② Measuring the quality of the constructed library: Measure the concentration and detect the fragment size of the constructed library. It is required that the total DNA > 500 bp and the DNA fragment range is 300 - 500 bp.

[0041] ③ Capturing and sequencing: Mix the probe synthesized in S4 with the hybridization buffer and place it in a test tube. Perform a PCR reaction and hybridization at 65°C for 16 h. Then, selectively capture the target fragment. Use the library amplification primer and high-fidelity DNA polymerase to amplify the captured target fragment. Then, perform two rounds of purification to ensure the purity of the library. Finally, use Qubit and qPCR technologies to measure the concentration of the purified library and perform sequencing using PE150 on the MGISEQ-2000 sequencing platform.

[0042] ④ Determining the final probe: Further evaluate the obtained SNP sites. The evaluation indicators include a site deletion rate ≤ 10%, a heterozygous mutation rate ≤ 30%, and a MAF ≥ 0.01. Based on the test samples, delete the sites with low capture efficiency among the selected sites, and determine and retain the final effective and available sites.

[0043] Based on the above steps, a total of 61,566 SNP sites were obtained, which is the final 60K chip for the main commercial pig breeds based on targeted capture sequencing. The distribution of SNP markers is as Figure 2 shown, among whichFigure 2 In which, a represents the density map of SNPs on each chromosome, Figure 2 b represents the number of SNPs on different chromosomes, Figure 2 c represents the SNP spacing on different chromosomes, Figure 2 d represents the SNP MAF distribution of different breeds. It can be seen from the figure that the chip is basically designed according to the design principle, the SNP distribution is relatively uniform, and the SNP distribution on each chromosome is also relatively uniform, meeting the expectations of chip design. The minor allele frequency is mainly concentrated above 0.15, and the MAF meets the chip design requirements. The distribution of SNP markers in the gene structure is as Figure 3 shown, mainly concentrated in introns and intergenic regions, consistent with the distribution law of genes on chromosomes, further indicating that the distribution of chip marker sites is uniform and reasonable.

[0044] Example 2: Quality evaluation of 60K liquid chip genotyping A total of 1443 pigs of major commercial pig breeds were randomly selected, including 363 Duroc pigs, 655 Landrace pigs, and 425 Large White pigs. The 60K chip prepared in Example 1 above was used for genotyping detection, and the genotyping effect was evaluated for quality. The evaluation results are as follows: S1. Detection rate: The capture efficiency of sample sites is an important indicator to measure the quality of the chip. As Figure 4 shown in a, the SNP detection rates of the three breeds are all above 99%. Among them, the average detection rate of Duroc pig samples is 99.53%, the average detection rate of Landrace pig samples is 99.45%, the average detection rate of Large White pig samples is 99.59%, and the average detection rate of all samples of the three breeds is 99.51%. The above results indicate that the chip has a very good detection effect.

[0045] S2. Stability: The detection stability of the chip is another important indicator to measure the quality of the chip, generally measured by the consistency of the genotype detection results of repeated samples twice. Randomly select 10 samples and use the 60K chip prepared in Example 1 above to perform repeated detection on them. The results are as Figure 4 shown in b. The genotype detection consistency of the 10 repeated individuals is relatively high, specifically between 99.37% and 99.74%, with an average of 99.69%, indicating that the detection stability of the chip is very good.

[0046] S3. Principal component analysis (PCA): To ensure breed compatibility, 1000 breed-specific SNPs were added to each breed during the preparation process of Example 1 above. To detect whether the chip can be used for genetic structure division, 200 Duroc, Landrace, and Large White pigs were randomly selected for genotype detection, and PCA was used to divide the chip data by breed. As Figure 5 shown, the chip can clearly separate the population structures of the three breeds.

[0047] Example 3: Compatibility Evaluation of Different Chips With the development of sequencing technology, chips are being updated rapidly. The medium-density chips for pigs have also gone through several iterations, such as from the commonly used solid-phase GGP50K to KPSPBC50K, and then to GBTS50K, etc. The replacement of chips has made sample detection cheaper, customization more flexible, and detection speed faster. However, the compatibility of different chips has also become an important factor in evaluating chip quality. Generally, the genotype imputation effect of different chips is used as an indicator to measure chip compatibility. The genotype imputation effect analysis was carried out on the chip prepared in Example 1 above and three other commonly used medium-density chips (GGP50K, KPSPBC50K, GBTS50K) and sequencing data to solve the problem of joint analysis of different chips, as follows: S1. Data collection: Based on the sequencing data filled with 5928 samples in Example 1 above, 1000 pigs of each of Landrace, Large White, and Duroc were randomly selected as reference groups of different breeds, and another 200 pigs of each of Landrace, Large White, and Duroc were randomly selected as validation groups. The genotype data of the validation groups were respectively extracted into the loci of the autosomes of different chips. The coincidence of the autosome loci of the chip prepared in Example 1 with GGP50K, KPSPBC50K, and GBTS50K is as Figure 6 shown. This chip has the largest number of coincident autosome loci with GBTS50K and the smallest number of coincident loci with KPSPBC50K.

[0048] S2. Analysis of the imputation effect of chips on each other: Use the Beagle v5.4 software to impute the autosome loci of different chips with each other, and calculate the correlation coefficient between the imputed genotype and the original genotype. As Figure 7 shown, the accuracy of imputing different chips to the 60K chip or the 60K chip to different chips is relatively high, indicating that the compatibility of this chip with the other three chips is good.

[0049] S3. Evaluation of the accuracy of imputing chips to sequencing data: Use the Beagle v5.4 software to impute the autosome loci of different chips to sequencing data. As Figure 8 shown, the accuracy of imputing the four chips to sequencing data is relatively high, and the accuracy of imputing the 60K chip prepared in Example 1 above to sequencing data is even higher.

[0050] Example 4: Evaluation of the Application Effect of 60K Liquid Chip in Genomic Selection Genomic selection is a new technology and method of milestone significance in animal breeding. In this example, the genomic selection effect analysis was carried out on the 60K liquid chip prepared in Example 1. The steps are as follows: S1. Sample and phenotypic data collection: The data used in this example came from samples of 1,419 pigs from two breeding farms, Hongxing in Heze, Shandong and Huayu in Binzhou, Shandong, including 356 Duroc pigs, 650 Landrace pigs, and 413 Large White pigs. Four production traits, namely, age at 100 kg (AGE), backfat thickness (BF), birth weight (BW), and total number of teats (TN), were collected.

[0051] S2. Genotype detection and quality control: The 60K liquid chip prepared in Example 1 was used to detect the genotypes of 1,419 individuals with phenotypic traits. The quality control criteria were as follows: ① Remove sex chromosome loci; ② Remove SNPs with a detection rate lower than 90%; ③ Remove SNPs with a minor allele frequency (MAF) lower than 0.05; ④ Remove individuals with a detection rate lower than 90%; ⑤ Remove loci that do not conform to the Hardy-Weinberg equilibrium.

[0052] S3. Evaluation of genomic selection accuracy: The DMU was used to estimate the genetic parameters of traits, and the GBLUP model was used to estimate the genomic breeding values. The genomic selection accuracy was the correlation coefficient between the genomic breeding values (GEBV) and the breeding values (EBV) of the validation population. As Figure 9 shown, the genomic selection accuracy met the expectations. The genomic selection accuracies of the same trait in different breeds were different, which was related to the differences in the breeds themselves. This chip can be used for genomic selection breeding of major commercial pig breeds.

[0053] Although the present invention has been illustrated and described with reference to specific embodiments, many other changes can be made without departing from the principles of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a commercial pig breed 60K liquid phase chip based on targeted capture sequencing, characterized in that: The following steps are involved: S1: SNP site screening: Based on the whole genome resequencing data of the target population, combined with known chip sites and known economic trait functional sites of pigs, SNP sites are screened on the basis of ensuring uniform distribution on each chromosome; S2: Probe design: Design and synthesize liquid phase probes based on the SNP sites screened in step S1, where the synthesized probes must meet the following conditions: probe length is 105-115bp (e.g. 110bp); the GC content of the SNP site is controlled between 30% and 70% to ensure stability and hybridization efficiency; the GC content of the functional site is controlled between 20% and 80%; the number of homologous regions cannot be more than 5, and the selected regions avoid simple sequence duplications and blank regions to reduce nonspecific binding; design two nucleotide sequences with 60%-70% overlap covering the SNP site for the screened SNP site to ensure site capture; S3: Probe synthesis: Synthesize two DNA nucleotide sequences with a length of 105-115 bp (e.g., 110 bp) and a biotin group modified at the 5' end according to the nucleotide sequence designed in the above requirements; The synthesized porcine chip probes are mixed in equal molar mass and diluted to a final concentration of 1-5 pmol / mL (e.g., 3 pmol / mL) using EDTA and Tris-HCl buffer to prepare a preliminary chip probe mixture; S4: Determine the final probe: Use the test sample to perform probe detection, delete the sites with low capture efficiency based on the test sample data, determine and retain the final valid and available sites, which is the pig 60K liquid phase chip.

2. The method for preparing a commercial pig breed 60K liquid phase chip according to claim 1, characterized in that: The specific steps of S1 SNP site screening are as follows: ① Collect samples of the target population for whole genome resequencing to obtain genome variation information of the target population; ② Select common sites with MAF>0.1 in the target population from the existing pig commonly used medium density chips as the skeleton of the designed sites, and fill the background sites in the areas outside the common sites; ③ Add breed-specific SNP sites to each breed (for example, select 500-1500, such as 1000); ④ Add functional sites closely related to pig production traits; ⑤ Add the remaining sites to 55-65K to ensure uniform distribution, avoid linkage sites, and avoid complex areas of the genome.

3. The method for preparing a commercial pig breed 60K liquid phase chip according to claim 1, characterized in that: The specific steps of S4 determining the final probe are as follows: ① extracting the DNA of the pig samples to be tested to build a library, specifically extracting the DNA of the pigs to be tested and constructing a pig DNA high-throughput sequencing library; ② determining the quality of the library construction, specifically using Qubit2.0Eluorometer for quantitative analysis, and using agarose gel electrophoresis to detect the quality of the library fragments to ensure that DNA>500bp, and the DNA fragment range is 300-500bp; ③ capturing sequencing, specifically mixing the probe with the hybridization buffer and placing it in a test tube for PCR reaction, and then amplifying the captured target fragments using library amplification primers and high-fidelity DNA polymerase, and then performing two rounds of purification, and finally using Qubit and qPCR technology to determine the concentration of the purified library, and sequencing on the MGISEQ-2000 sequencing platform; ④ determining the final probe and site set, specifically further evaluating the SNP sites according to the capture of the test samples, the evaluation indicators include site deletion rate ≤10%, heterozygous mutation rate ≤30%, MAF≥0.01, deleting sites with low capture efficiency in the selected sites, and determining and retaining the final valid and available sites.

4. The commercial pig breed 60K liquid phase chip obtained by the commercial pig breed 60K liquid phase chip preparation method as described in claim 1, 2 or 3.

5. The method for performing pig genome typing using the commercial pig breed 60K liquid phase chip as claimed in claim 4, characterized in that: The steps include: S1. Construction of pig DNA high-throughput sequencing library: Extract the genomic DNA of the pig to be tested and fragment it to generate DNA fragments suitable for high-throughput sequencing; complete the construction of high-quality sequencing library by adding sequencing adapters and performing end repair, and perform quality testing on the library to ensure that it meets the sequencing requirements; S2. Capturing target genomic fragments: combining the commercial pig breed 60K liquid phase chip prepared in advance as claimed in claim 3 with the pig DNA sequencing library constructed in step S1, and using the probes in the chip to hybridize and bind with the target genomic fragments; by using a magnetic bead separation system, the DNA fragments containing the target SNP sites in the library are targeted for capture; After the washing step, non-target DNA fragments are removed, leaving only the DNA in the target region; S3, amplification, purification and high-throughput sequencing: The target DNA fragment captured in step S2 is amplified by PCR, the amplified product is purified, and sequenced by a high-throughput sequencing platform; S4. Typing results: After processing and comparison of the data generated by sequencing, the precise SNP typing results of the pig genome are finally obtained.

6. The method according to claim 5, characterized in that The specific method for constructing a pig DNA high-throughput sequencing library in step S1 is: extract the genomic DNA of the pig to be tested and physically break it to generate DNA fragments of uniform length; add suitable sequencing adapters to the fragments through end repair and A-tail addition steps, and then construct a high-throughput sequencing library; the quality of the library is evaluated by precision testing equipment to ensure the success of subsequent capture and sequencing.

7. The method according to claim 5, characterized in that The capture method of step S2 is: combining the constructed pig DNA high-throughput sequencing library with the commercial pig breed 60K liquid phase chip; the probes in the chip bind to specific genomic fragments through hybridization, and then the streptavidin-biotin system is used to capture the target fragments onto magnetic beads.

8. The method according to claim 5, characterized in that After the sequencing data in step S3 are quality controlled, they are analyzed using professional bioinformatics software, and the resulting reads are aligned to the porcine reference genome for genotyping and variation detection.

9. The method according to any one of claims 4 to 8, characterized in that The pig genome typing is for the genome typing of Landrace pigs, Large White pigs or Duroc pigs.

10. Application of the commercial pig breed 60K liquid phase chip as claimed in claim 3 in pig genome typing, pig genetic diversity evaluation, pig breed identification, pig genome selection, genome-wide association analysis or pig genetic improvement; Preferably, it is used for screening and breeding and / or genetic improvement of target traits in Landrace, Large White or Duroc pigs.

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