Liquid phase chip for identifying pig breeds as well as preparation method and application of liquid phase chip

Abstract

The invention relates to a liquid phase chip for identifying pig breeds and a preparation method and application thereof, and the liquid phase chip for identifying pig breeds comprises a set of nucleotide probes for detecting 51 and 188 SNP (Single Nucleotide Polymorphism) sites. The invention further discloses a preparation method of the liquid chip for identifying the pig breeds. The invention further comprises application of the liquid chip in pig genetic typing and Yunnan local pig breed distinguishing. The liquid phase chip is provided with 51 and 188 DNA double-chain capture probes in total, the DNA double-chain capture probes comprise specific sites of a plurality of Yunnan local pig breeds such as southern Yunnan small-ear pigs, Shandai pigs and Wujin pigs and specific sites of foreign introduced breeds such as Duroc and Berkshire, 51 and 188 high-quality SNP sites are screened out in total, and 51 and 165 sequence intervals can be covered. According to the method, germplasm improvement is carried out according to important economic characters of Yunnan local pig breeds, early selection is carried out, the genetic process is accelerated, the selection period is shortened, and the breeding efficiency and accuracy of the Yunnan local pig breeds are improved.

Description

Technical Field

[0001] The present invention relates to a liquid phase chip for identifying pig breeds, a preparation method and application thereof, and in particular to a 50k liquid phase chip for Yunnan local breeds and cultivated pig breeds, a preparation method and application thereof. Background Art

[0002] Single nucleotide polymorphism (SNP) refers to nucleic acid sequence diversity caused by changes in a single nucleotide within a chromosome genome and is a ubiquitous form of genetic variation. SNPs are genetically stable and widely distributed and densely distributed within individual genomes. This property allows them to effectively avoid the influence of morphological and environmental factors, significantly accelerating breeding processes. It is considered the third generation of molecular marker technology.

[0003] With the continuous advancement of modern molecular breeding technologies, whole-genome high-throughput sequencing has become the primary method for SNP typing. For species with a reference genome sequence, researchers typically design SNP microarrays using this known sequence information. The technical principle involves randomly truncating the genomic sequence to allow for hybridization with the specific nucleotide sequences of the microarray probes, forming double strands. This allows for identification and detection of SNP genotypes at the corresponding loci. Currently, gene chip probe carriers are primarily categorized into two types: solid-phase and liquid-phase. Solid-phase microarrays rely on spatial addressing to identify different array elements, with reactions occurring on a solid substrate surface. Liquid-phase microarrays, on the other hand, identify array elements based on physical and optical signals (such as size or color) of microspheres. Both offer advantages such as high efficiency, high sensitivity, and high throughput. However, compared to solid-phase microarrays, liquid-phase microarrays allow for tailored probe labeling based on the species and eliminate the need for washing steps, making them simpler, more flexible, and less expensive to use. Consequently, they have gained widespread application in genomics, proteomics, disease diagnostics, and SNP research.

[0004] With the development of high-throughput technology, researchers have developed various types of liquid microarrays to meet diverse research needs. Currently, liquid microarrays designed for SNP functional loci in commercial pigs are relatively mature, such as the 50K SNP liquid microarray designed by Ding Xiangdong et al. and the 80K functional locus gene chip designed by Zhao Shuhong et al. While these microarrays can rapidly detect SNP loci for economic and reproductive traits in commercial pigs, they lack consideration for the genetic diversity of local pig breeds, resulting in poor inter-breed and local breed applicability.

[0005] Yunnan Province, with its ecological and geographical diversity, has nurtured a rich resource of endemic pig breeds, such as the Diannan Small-Eared Pig, the Saba Pig, and the Wujin Pig. These endemic pig breeds not only possess distinct characteristics but also offer advantages in meat quality, stress resistance, and tolerance to roughage, making them highly favored by local consumers. In recent years, with the increasing demand for high-quality meat products, the market demand for premium pork has continued to rise. However, compared with commercial pig breeds, Yunnan endemic pig breeds, while offering superior meat quality, generally suffer from low reproductive efficiency and poor growth performance, resulting in high breeding and production costs. Furthermore, weak basic research on endemic pig breeding, a lack of systematic research on germplasm characteristics, and a lack of continuous breeding efforts have severely hampered the utilization of endemic pig germplasm resources. Therefore, there is an urgent need to develop a high-density SNP array to improve the germplasm of Yunnan endemic pig breeds for key economic traits and conduct early selection to accelerate genetic progression, shorten the selection cycle, and improve the breeding efficiency and precision of Yunnan endemic pig breeds. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a liquid phase chip for identifying pig breeds and its preparation method and application, so as to carry out early selection for Yunnan local breeds and cultivated pig breeds to accelerate the genetic process, shorten the selection cycle, and improve the breeding efficiency and accuracy of Yunnan local pig breeds.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] A liquid phase chip for identifying pig breeds includes a set of nucleotide probes for detecting 51,188 SNP sites, as shown in Table 1:

[0009] Table 1 SNP site information

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[0295] The value before the “:” symbol in the table is the chromosome number, and the value after the “:” symbol is the position of the SNP site on the chromosome; the SNP sites in the table are arranged in order from left to right and from top to bottom by chromosome and site.

[0296] The present invention also provides a whole genome liquid phase chip for detecting the SNP marker combination.

[0297] The present invention also provides a kit for identifying the SNP molecular marker combination, which comprises the liquid phase chip for identifying pig breeds.

[0298] The present invention also provides a method applicable to pig SNP screening, which uses a kit comprising the SNP molecular marker combination.

[0299] The present invention also provides an application method of the pig SNP molecular marker combination, liquid phase chip and related kit.

[0300] The method for preparing a liquid phase chip for identifying pig breeds of the present invention includes the following steps:

[0301] 1. Extraction of DNA from animals;

[0302] 2. DNA quality inspection;

[0303] 3. Genomic library construction and alignment;

[0304] 4. Analysis of population genetic structure;

[0305] 5. Chip site screening;

[0306] The liquid phase array site selection was performed through three aspects: resequencing, important functional sites, and variety-specific sites. The specific implementation methods are as follows:

[0307] (1) Candidate site screening: Calculate the quality indicators of the resequencing sites and select SNP polymorphic sites that meet the requirements of heterozygosity ≤ 0.4, site deletion frequency < 0.1, MAF ≥ 0.1, and sequencing depth ≥ 10x in all samples as candidate sites;

[0308] (2) adding SNPs that have been reported to be associated with important economic and pathogenic traits in pigs to the backbone loci;

[0309] (3) Selection of breed-specific loci was performed to distinguish Yunnan local breeds. The specific method of loci selection was as follows: Fst values ​​between SNP loci of three Yunnan local pig breeds and other pig breeds were calculated using VCFtools (v 0.1.13). Loci with Fst>0.5 were selected to screen the population quality according to the principle of uniform density distribution. The indicators all met the requirements of minimum allele frequency (MAF) ≥ 0.05, deletion rate < 0.1, and sequencing depth ≥ 8X.

[0310] (4) Site probe design and screening criteria: Extract 100 bp of sequences upstream and downstream of the SNP site, analyze its specificity and GC content, and remove low-quality probes such as probes containing repetitive sequences in the flanking sequences;

[0311] (5) Site density screening;

[0312] 6. Chip site annotation;

[0313] 7. Preparation of Yunnan pig 50K liquid phase chip

[0314] The sites were integrated according to the above method. After the sites were determined, probes were designed and synthesized for the sites. The probe design was based on the following criteria:

[0315] (1) The probe sequence length is 100 bp;

[0316] (2) the copy number of the whole genome does not exceed 5;

[0317] The probe does not contain N bases and has a GC content ranging from 25% to 75%, preferably a probe with a GC content close to 50%.

[0318] The method of the present invention for genotyping using a liquid phase chip for identifying pig breeds comprises the following steps:

[0319] 1. Sample collection;

[0320] 2. DNA extraction and quality control;

[0321] 3. cGPS library construction and quality control

[0322] The DNA after quality control is used for library construction. After completing library construction and quality inspection, the library is sequenced on the BGI high-throughput sequencing platform. The specific experimental operation process is as follows:

[0323] (1) Use fragmentase to digest the DNA sample, repair the digested ends, and add an A base to the 3' end. Use agarose gel electrophoresis to detect the fragment size. The gDNA interrupted by enzyme digestion has no main band and the fragment range is between 100-500bp.

[0324] (2) Use T4 ligase to connect the sequencing adapter to the DNA fragment, and purify the ligation product using purification magnetic beads. The fragment size of the purified product is detected by agarose gel electrophoresis. The purified fragment range is between 150-500 bp.

[0325] (3) The purified ligation products were amplified and enriched, and the amplified products were screened for fragments using purified magnetic beads. The concentration of the fragment-screened products was determined using a Qubit fluorescence quantification instrument, and the fragment size was determined by agarose gel electrophoresis. The library fragments were between 200 and 400 bp.

[0326] (4) Take 200 ng of the constructed library, concentrate the library, add the probe and hybridization reagent, and incubate at 50°C for 16-24 hours to complete the hybridization reaction.

[0327] (5) The target segment is captured using capture magnetic beads, and the captured product is washed with a cleaning solution to remove non-specific binding fragments. Then, a round of PCR amplification is performed, and the amplified product is purified using purification magnetic beads.

[0328] (6) The library concentration was determined using a Qubit fluorescence quantification instrument, and the fragment size was determined by agarose gel electrophoresis. The library fragments were between 200 and 400 bp. Once the concentration and fragment size were qualified, the cGPS sequencing library construction was completed. The prepared library was sequenced using the MGI high-throughput sequencing platform with a sequencing read length of PE150.

[0329] 4. Data Analysis

[0330] The raw data after high-throughput sequencing is processed by quality control filtering and other processes. The specific process is as follows:

[0331] (1) Use FASTP software to remove adapter fragments and low-quality reads to obtain high-quality CleanReads;

[0332] (2) Use BWA software to align the obtained clean reads with the reference genome (Sscrofa 11.1) and perform position sorting to obtain the sample sorted bam file;

[0333] (3) Use GATK software to analyze the variant sites of the sequencing results and obtain the genotyping results of the target sites.

[0334] The application of the liquid phase chip for identifying pig breeds in the present invention in pig genotyping comprises the following steps:

[0335] (1) Obtain DNA samples from the pigs to be tested;

[0336] (2) Obtain genotype data of the pigs to be tested;

[0337] (3) Compare genotype data with genomic data.

[0338] The application of the liquid phase chip for identifying pig breeds of the present invention in distinguishing Yunnan local pig breeds comprises the following steps:

[0339] (1) Obtain genotype data of the pigs to be tested;

[0340] (2) PLINK (v1.9) was used to extract 598 Fst loci from the genotype data of each sample;

[0341] (3) Use the FST algorithm to calculate the genetic distance matrix to reflect the degree of differentiation between populations;

[0342] (4) MEGA (v11) was used to convert the format of the genetic distance matrix, and the NJ tree was constructed and the Bootstrap test was performed to finally distinguish the Yunnan small-eared pig, Wujin pig and Saba pig.

[0343] The liquid phase chip involved in the present invention has a total of 51,188 double-stranded DNA capture probes, including specific sites for multiple local Yunnan pig breeds such as the Yunnan Small-eared Pig, Saba Pig and Wujin Pig, as well as imported foreign breeds such as Duroc and Berkshire. A total of 51,188 high-quality SNP sites were screened out, covering 51,165 sequence intervals. The positions of the sites in the pig reference genome (Genome assembly Sscrofa11.1) are shown in Table 1.

[0344] The present invention improves the germplasm of important economic traits of Yunnan local pig breeds and carries out early selection to accelerate the genetic process, shorten the selection cycle, and improve the breeding efficiency and accuracy of Yunnan local pig breeds.

[0345] The present invention aims to develop a 50K liquid phase chip that can be widely used for Yunnan local breed pigs and general commercial pigs, and discloses the SNP probe design principles and the preparation and application methods of the liquid phase chip involved in the invention. The liquid phase capture probe involved in the invention contains multiple DNA sequence polymorphic sites of Yunnan local breeds, foreign pig breeds, and Dubadian hybrid pigs. The site coverage is high, the polymorphism is good, and the site distribution is uniform on the chromosome. The use of the present invention can realize low-cost and efficient detection of single nucleotide variations, making up for the shortcomings of existing technologies in the conservation and breeding of Yunnan local pig breeds. At the same time, it can also solve the problems of long breeding cycle, high cost and low efficiency in my country's commercial large-scale pig farms.

[0346] The present invention belongs to the field of genome chip technology. Based on the targeted capture sequencing technology of the second-generation sequencing technology, a 50K molecular marker combination that can be widely used in Yunnan local breed pigs and general commercial pigs, a liquid phase chip based on the combination, and its application method are designed and prepared. The SNP chip involved is composed of 51,188 highly specific and highly accurate single nucleotide polymorphism sites, including 49,768 sites evenly distributed on chromosomes, 822 sites significantly associated with important economic traits and disease resistance traits such as pig breeding, fattening, meat quality and growth, and 598 Fst sites for identifying Yunnan local breeds. Based on this molecular marker, the present invention has also developed a high-throughput, high-sensitivity liquid phase gene chip that can accurately and efficiently perform SNP genotyping on the tested samples, which can be widely used in many applications such as population genetic structure analysis, variety breeding and identification, and whole-genome association analysis.

[0347] The beneficial effects of the present invention include but are not limited to the following aspects:

[0348] 1. The present invention and the solutions involved therein not only refer to a number of high-quality local pig breeds in Yunnan, such as the small-eared pig in southern Yunnan, the Saba pig and the Wujin pig, but also take into account a large amount of resequencing data of introduced breeds such as Duroc and Berkshire, and select a total of 51,188 high-quality SNP sites. The average detection rate of SNPs on the chip is 99.12%, and the average genotype consistency of technical repeat samples is 99.10%, both showing the excellent stability and accuracy of the product. Through the precise and rapid SNP detection technology of the present invention, not only can the breeding problem of large-scale pig farms be solved, but it is also suitable for the mining of important economic trait genes of Yunnan local pig breeds and cultivated varieties, QTL positioning and whole-genome selection breeding, which can effectively improve the efficiency and accuracy of local excellent breed breeding, accelerate the seed conservation and breeding progress of Yunnan local pigs, and fully fill the technical gap of genomic selection breeding of Yunnan local pig breeds.

[0349] 2. The present invention and the liquid-phase chip involved are targeted sequencing technologies based on the second-generation sequencing technology. Compared with traditional resequencing or solid-phase chips, they can remove a large amount of background interference from invalid sites and accurately locate the required SNP sites. Technically, the invention is based on liquid-phase reaction kinetics, so the reaction speed is fast, the incubation time is shorter than traditional solid-phase detection, the operation is simple and fast, and the amount of sample required during the determination process is small, which is suitable for analyzing small-volume rare samples. In addition, the present invention also has strong specificity. The chip involved in the present invention can automatically distinguish between molecules bound to the microspheres and unbound molecules without washing, and only reads the fluorescent signal on a single microsphere, with high signal-to-noise ratio and repeatability. At the same time, the chip also has high flexibility. Users can add or subtract probe cross-linked microspheres and labeled molecules as needed to meet the needs of different detection items. BRIEF DESCRIPTION OF THE DRAWINGS

[0350] Figure 1 This is a PCA analysis diagram of the development population of the 50K liquid phase chip of the present invention;

[0351] Figure 2 This is a genetic structure analysis diagram of the development population of the 50K liquid phase chip of the present invention;

[0352] Figure 3 The density distribution diagram of the SNP sites included in the 50K liquid phase chip of the present invention on the chromosome;

[0353] Figure 4 This is a diagram showing the site detection results of genotyping 385 pigs using the liquid phase chip of the present invention;

[0354] Figure 5 This is a graph showing the genotype consistency rate of a single sample after two repeated typing using the liquid phase chip of the present invention;

[0355] Figure 6This is a comparison chart of the consistency between the genotypes obtained by liquid phase chip detection and the genotypes obtained by resequencing (n=20);

[0356] Figure 7 This is a phylogenetic tree analysis diagram of the breed-specific sites contained in the liquid phase chip of the present invention for the Diannan Small-ear Pig, Wujin Pig and Saba Pig;

[0357] Figure 8 This is a graph showing the results of GWAS association analysis based on the liquid phase chip of the present invention. DETAILED DESCRIPTION

[0358] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0359] For details not listed in the embodiment examples, the operation was carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments without manufacturer's name were all commercially available conventional products.

[0360] Example 1

[0361] Chip design process and preparation

[0362] Whole-genome resequencing and literature analysis were used to obtain high-quality SNP sites in the pig genome. The breeds and sample sizes of the samples used are shown in Table 2. The specific design and preparation process are described below:

[0363] Table 2 Number and origin of groups

[0364]

[0365] 1.1 DNA extraction from experimental animals

[0366] (2) Wash the ear margin tissue with physiological saline, remove excess hair, and place it in a clean enzyme-free centrifuge tube; place 3 grinding beads in each centrifuge tube, add 500 μL of DNA lysis buffer and 20 μL of proteinase K solution, and place it in a homogenizer to fully homogenize;

[0367] (3) After homogenization until no obvious lumps of tissue are observed by naked eye, place the mixture in a water bath shaker and bathe in 55°C water for 120-180 min until the tissue is completely lysed. Then, remove the mixture and place it on ice.

[0368] (4) Add 1 mL of anhydrous ethanol pre-cooled to 4°C to the supernatant and mix thoroughly. Centrifuge for 3 minutes to retain the nucleic acid precipitate.

[0369] (5) Remove the supernatant and add 1 mL of pre-cooled 75% ethanol solution. Shake thoroughly to mix to elute the salts. Centrifuge for 10 minutes and remove the supernatant.

[0370] (6) Repeat step (4);

[0371] (7) Aspirate the remaining liquid and place the centrifuge tube in a clean bench to dry until the nucleic acid precipitate is transparent;

[0372] (8) Add 50 μL of sterile enzyme-free water and vortex to dissolve. Centrifuge at 4000 rpm / min for 30 s. Place the extracted DNA solution on ice and prepare for quality inspection.

[0373] 1.2 DNA quality inspection

[0374] To ensure the quality of the library, the extracted DNA samples were tested for concentration, purity, and integrity in the following manner. Qualified samples were then used to construct the library.

[0375] (1) DNA fragment size and DNA integrity were detected by electrophoresis using 1.5% agarose gel at a voltage of 120 V for 30 min.

[0376] (2) The purity of the sample was tested using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). The OD 260 / 280 ratio was between 1.8 and 2.2, and there was no protein or visible impurities contamination.

[0377] (3) DNA concentration was measured using a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA). The concentration was greater than 50 ng / μl and the total amount was greater than 2 μg.

[0378] 1.3 Genomic library construction and alignment

[0379] After the DNA samples passed the test, the library was constructed using enzyme digestion. The Agilent 2100 / 2200 Bioanalyzer was used to check whether the fragment size of the library was between 300 and 500 bp. The effective concentration of the library was accurately quantified using Q-PCR. To ensure the quality of the library, the qualified library was sequenced using the BGI sequencing platform PE150. After the library was constructed, the raw data was quality controlled to obtain clean reads. The specific standards are as follows:

[0380] (1) Use Sentieon (v 202503) to align reads to the porcine reference genome (Sscrofa11.1), perform position sorting, and mark duplicate reads;

[0381] (2) Sentieon detects variant sites in each sample and obtains the gVCF of each sample;

[0382] (3) Use Sentieon to perform joint-calling and jointly analyze the gVCF of all samples to obtain the variation results of each individual in the population;

[0383] (4) To ensure the accuracy of SNPs, the SNP sites obtained after the joint analysis were initially hard filtered, and the filtering criteria were as follows: QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0.

[0384] 1.4 Analysis of population genetic structure

[0385] To ensure the reliability of the resequencing data, we used it to analyze and verify the population genetic structure. The specific implementation method is as follows: Principal components analysis (PCA) was performed using PLINK (v 1.9) and visualized using R (v4.2.1). The development population of the chip can be clearly separated, and the individuals within the population are clustered relatively tightly, indicating that there is no obvious confounding in the development population (see Figure 1 , Figure 1 This is a PCA analysis diagram of the development population of the 50K liquid phase chip of the present invention); phylogenetic tree analysis was performed on 126 resequencing individuals. The VCF2Dis (v 1.47) (https: / / github.com / BGI-shenzhen / VCF2Dis) program was used to convert the variation call format (VCF) file into a relationship matrix. FastME (v 2.0) provides a distance algorithm to infer the phylogenetic relationship between varieties. Finally, iTOL (v 6.7.4) was used to visualize the phylogenetic relationship between chip construction populations. The population phylogenetic tree diagram can clearly illustrate the relationship between the development populations, and there is no obvious consanguinity between the populations (see Figure 2 , Figure 2 (This is a genetic structure analysis diagram of the development population of the 50K liquid phase chip of the present invention).

[0386] 1.5 Chip site screening

[0387] The liquid phase array site selection was performed through three aspects: resequencing, important functional sites, and variety-specific sites. The specific implementation methods are as follows:

[0388] (1) Candidate site screening: Calculate the quality indicators of the resequencing sites and select SNP polymorphic sites that meet the requirements of heterozygosity ≤ 0.4, site deletion frequency < 0.1, MAF ≥ 0.1, and sequencing depth ≥ 10x in all samples as candidate sites;

[0389] (2) adding SNPs that have been reported to be associated with important economic and pathogenic traits in pigs to the backbone loci;

[0390] (3) Selection of breed-specific loci was performed to distinguish Yunnan local breeds. The specific method of loci selection was as follows: Fst values ​​between SNP loci of three Yunnan local pig breeds and other pig breeds were calculated using VCFtools (v 0.1.13). Loci with Fst>0.5 were selected to screen the population quality according to the principle of uniform density distribution. The indicators all met the requirements of minimum allele frequency (MAF) ≥ 0.05, deletion rate < 0.1, and sequencing depth ≥ 8X.

[0391] (4) Site probe design and screening criteria: Extract 100 bp of sequences upstream and downstream of the SNP site, analyze its specificity and GC content, and remove low-quality probes such as probes containing repetitive sequences in the flanking sequences;

[0392] (5) Site density screening: All sites were merged and the evenly distributed SNP sites were selected as the whole genome background sites according to the principle of evenly distributed physical positions of the sites on the genome. The average spacing between sites was 47.5 Kb. The final distribution of sites on the pig chromosomes was as follows: Figure 3 As shown, Figure 3 This is the density distribution diagram of the SNP sites contained in the 50K liquid phase chip of the present invention on the chromosome. It can be seen that the chip sites are evenly distributed on the chromosome and the density design is reasonable.

[0393] 1.6 Microarray Site Annotation

[0394] After site screening, Bedtools software (v2.30.0) was used to perform functional annotation of the chip sites with reference to the pig QTL database (https: / / www.animalgenome.org / cgi-bin / QTLdb / SS / index), and a total of 6,514 functional sites were obtained (as shown in Table 3).

[0395] Table 3 Classification and quantity of main economic traits of chips

[0396]

[0397]

[0398] 1.7 Preparation of Yunnan pig 50K liquid phase microarray

[0399] The site was integrated according to the above method, and the location of the site after integration on the pig genome is as follows Figure 6 As shown, Figure 6This is a comparison chart of the consistency between the genotypes obtained by liquid-phase chip detection and the genotypes obtained by resequencing. After the site is determined, probes are designed and synthesized for the site. Probe design is based on the following criteria:

[0400] (3) The probe sequence length is 100 bp;

[0401] (4) The copy number of the whole genome does not exceed 5;

[0402] (5) The probe does not contain N bases and has a GC content ranging from 25% to 75%, preferably a probe with a GC content close to 50%.

[0403] Example 2

[0404] The method for performing genotype detection using the liquid phase chip developed in Example 1 comprises the following steps:

[0405] 1. Sample collection

[0406] 385 healthy pigs were randomly selected from the breeding farm, and their ear margin tissues were collected and immediately placed in 75% ethanol and stored in a -80℃ refrigerator for future use.

[0407] 2. DNA extraction and quality control

[0408] The collected samples were subjected to DNA extraction using a magnetic bead method. The extracted DNA samples were quality checked according to the method used in Example 1. The quality checked samples were stored at -20°C for future use.

[0409] 3. cGPS library construction and quality control

[0410] After step 2, the DNA is quality-controlled for library construction. After completing library construction and quality control, the library is sequenced using the BGI high-throughput sequencing platform. The specific experimental operation process is as follows:

[0411] (1) Use fragmentase to digest the DNA sample, repair the digested ends, and add an A base to the 3' end. Use agarose gel electrophoresis to detect the fragment size. The gDNA interrupted by enzyme digestion has no main band and the fragment range is between 100-500bp.

[0412] (2) Use T4 ligase to connect the sequencing adapter to the DNA fragment, and purify the ligation product using purification magnetic beads. The fragment size of the purified product is detected by agarose gel electrophoresis. The purified fragment range is between 150-500 bp.

[0413] (3) The purified ligation products were amplified and enriched, and the amplified products were screened for fragments using purified magnetic beads. The concentration of the fragment-screened products was determined using a Qubit fluorescence quantification instrument, and the fragment size was determined by agarose gel electrophoresis. The library fragments were between 200 and 400 bp.

[0414] (4) Take 200 ng of the constructed library, concentrate the library, add the probe and hybridization reagent, and incubate at 50°C for 16-24 hours to complete the hybridization reaction.

[0415] (5) The target segment is captured using capture magnetic beads, and the captured product is washed with a cleaning solution to remove non-specific binding fragments. Then, a round of PCR amplification is performed, and the amplified product is purified using purification magnetic beads.

[0416] (6) The library concentration was determined using a Qubit fluorescence quantification instrument, and the fragment size was determined by agarose gel electrophoresis. The library fragments were between 200 and 400 bp. Once the concentration and fragment size were qualified, the cGPS sequencing library construction was completed. The prepared library was sequenced using the MGI high-throughput sequencing platform with a sequencing read length of PE150.

[0417] 4. Data Analysis

[0418] The raw data after high-throughput sequencing is processed by quality control filtering and other processes. The specific process is as follows:

[0419] (1) Use FASTP software to remove adapter fragments and low-quality reads to obtain high-quality CleanReads;

[0420] (2) Use BWA software to align the obtained clean reads with the reference genome (Sscrofa 11.1) and perform position sorting to obtain the sample sorted bam file;

[0421] (3) Use GATK software to analyze the variant sites of the sequencing results and obtain the genotyping results of the target sites.

[0422] Example 3 Application of the developed liquid phase chip in pig genotyping

[0423] The application of the liquid phase chip developed in Example 1 in pig breed identification includes the following steps:

[0424] (1) Obtain DNA samples from 385 pigs to be tested;

[0425] (2) Obtaining genotype data of the pigs to be tested according to the method described in Example 2;

[0426] (3) The proportion of genotype data successfully matched to the genome, that is, the site detection rate was 98.92% to 99.51%, with an average of 99.31%. The site detection rate of most individuals was above 99.2% (e.g. Figure 4 As shown, Figure 4The results of the liquid phase chip of the present invention are shown in Figure 1. 20 samples were randomly selected for site consistency comparison, and the accuracy of the chip was evaluated by comparing the consistency of the results. Two of the samples were tested for site consistency using the chip typing technique, and the genotype consistency rates were 99.04% and 99.16% respectively (Figure 1). Figure 5 As shown, Figure 5 The results of the genotype consistency rate of a single sample using the liquid phase chip of the present invention for two repeated typing are shown in Figure 3. The chip typing results of 20 samples were compared with the resequencing data for site comparison. The results showed that the consistency of the 20 samples was 95.09% to 96.12%, with an average consistency of 96.16% (as shown in Figure 3). Figure 6 As shown, Figure 6 (Figure 3) shows the consistency comparison between the genotypes detected by the liquid phase chip of the present invention and the genotypes obtained by resequencing, which further illustrates the stability of the chip.

[0427] Example 4 Application of the developed liquid phase chip in distinguishing Yunnan local pig breeds

[0428] (1) Obtain the genotype data of the pigs to be tested according to the method described in Example 2

[0429] (2) PLINK (v1.9) was used to extract 598 Fst loci from the genotype data of each sample;

[0430] (3) Use the FST algorithm to calculate the genetic distance matrix to reflect the degree of differentiation between populations;

[0431] (4) MEGA (v11) was used to convert the format of the genetic distance matrix, and the NJ tree was constructed and the Bootstrap test was performed to finally distinguish the Yunnan small-eared pig, Wujin pig and Saba pig ( Figure 8 , Figure 8 (Figure 2 is the result of GWAS association analysis based on the liquid phase chip of the present invention).

[0432] Example 5 Application of the developed liquid phase chip in genome-wide association analysis

[0433] (1) Obtaining genotype data of the pigs to be tested according to the method described in Example 2;

[0434] (2) GEMMA (v0.98.5) software was used for trait association analysis, and the model was a linear mixed model (LMM);

[0435] (3) To control the multiple testing error, this study used the -indep-pairwise 50100.2 parameter of PLINK (v 1.9) to perform linkage disequilibrium analysis for SNP screening. Based on this, the Bonferroni multiple testing correction method was used to determine the significance threshold: the genome-wide significance level was set to 0.05 / N (N = the number of SNPs after quality control); multiple candidate SNP sites associated with economic traits (such as Figure 7 As shown, Figure 7 (This is a phylogenetic tree analysis diagram of the breed-specific sites contained in the liquid chip of the present invention for the Yunnan Small-ear Pig, Wujin Pig and Saba Pig).

Claims

1. A liquid phase chip for identifying pig breeds, characterized in that: Includes a set of nucleotide probes for detecting 51,188 SNP sites, as shown in Table 1: Table 1 SNP site information 2. The method for preparing the liquid phase chip for identifying pig breeds according to claim 1, characterized in that: The following steps are involved: (1) DNA extraction from animals; (2) DNA quality inspection; (3) Genomic library construction and alignment; (4) Analysis of population genetic structure; (5) Chip site screening.

3. The method for preparing a liquid phase chip for identifying pig breeds according to claim 2, wherein: Step (5) chip site screening, which involves selecting sites for the liquid phase chip through three aspects: resequencing, important functional sites, and variety-specific sites. Specifically, the following steps are included: (1) Candidate site screening: Calculate the quality indicators of the resequencing sites and select SNP polymorphic sites that meet the requirements of heterozygosity ≤ 0.4, site deletion frequency < 0.1, MAF ≥ 0.1, and sequencing depth ≥ 10x in all samples as candidate sites; (2) adding SNPs that have been reported to be associated with important economic and pathogenic traits in pigs to the backbone loci; (3) Select variety-specific loci to distinguish Yunnan local varieties; (4) Site probe design and screening criteria: Extract 100 bp of sequences upstream and downstream of the SNP site, analyze its specificity and GC content, and remove low-quality probes such as probes containing repetitive sequences in the flanking sequences; (5) Site density screening; (6) Chip site annotation; (7) Preparation of Yunnan pig 50K liquid phase chip.

4. The use of the liquid phase chip for identifying pig breeds according to claim 1, comprising a method for performing genotype detection, characterized in that: The steps are as follows: (1) Sample collection; (2) DNA extraction and quality control; (3) cGPS library construction and quality control; (4) Data analysis.

5. The use of the liquid phase chip for identifying pig breeds according to claim 4, characterized in that: The specific operation process of step (3) is as follows: (1) Use fragmentase to digest the DNA sample, repair the digested ends, and add an A base to the 3' end. Use agarose gel electrophoresis to detect the fragment size. The gDNA fragments cut by enzyme digestion have no main band and the fragment range is between 100-500 bp. (2) Use T4 ligase to connect the sequencing adapter and the DNA fragment, and purify the ligation product using purification magnetic beads; the fragment size of the purified product is detected by agarose gel electrophoresis, and the fragment size range after purification is between 150-500 bp; (3) The purified ligation products were amplified and enriched, and the amplified products were screened for fragments using purified magnetic beads; the concentration of the fragments after fragment screening was detected using a Qubit fluorescence quantification instrument, and the fragment size was detected by agarose gel electrophoresis. The library fragments were between 200 and 400 bp; (4) Take 200 ng of the constructed library, concentrate the library, and add probes and hybridization reagents; Incubate to complete the hybridization reaction; (5) Capturing the target segment using capture magnetic beads, washing the captured product with a cleaning solution to remove non-specific binding fragments, performing another round of PCR amplification, and purifying the amplified product using purification magnetic beads; (6) The library concentration was detected by Qubit fluorescence quantification instrument, and the fragment size was detected by agarose gel electrophoresis. The library fragments were between 200-400 bp. Once the concentration and fragment size were qualified, the cGPS sequencing library construction was completed. The prepared library was sequenced on the MGI high-throughput sequencing platform, and the sequencing read length was PE150.

6. The use of the liquid phase chip for identifying pig breeds according to claim 4, comprising a method for performing genotype detection, characterized in that: Step (4) Data analysis: quality control and filtering of the raw data after high-throughput sequencing. The specific process is as follows: (1) Use FASTP software to remove adapter fragments and low-quality reads to obtain high-quality Clean Reads; (2) Use BWA software to align the obtained clean reads with the reference genome and perform position sorting to obtain the sample sorted bam file; (3) Use GATK software to analyze the variant sites of the sequencing results and obtain the genotyping results of the target sites.

7. The use of the liquid phase chip for identifying pig breeds according to claim 1, including use in pig genotyping, characterized in that: The steps are as follows: (1) Obtain DNA samples from the pigs to be tested; (2) Obtain genotype data of the pigs to be tested; (3) Compare genotype data with genomic data.

8. The use of the liquid phase chip for identifying pig breeds according to claim 1, including use in distinguishing local pig breeds in Yunnan, characterized in that: The steps are as follows: (1) Obtain genotype data of the pigs to be tested; (2) Using PLINK to extract 598 Fst loci from the genotype data of each sample; (3) Use the FST algorithm to calculate the genetic distance matrix to reflect the degree of differentiation between populations; (4) MEGA was used to convert the format of the genetic distance matrix, and the NJ tree was constructed and the Bootstrap test was performed to finally distinguish the Yunnan small-eared pig, Wujin pig and Saba pig.

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