Min pig whole genome liquid phase chip and application thereof

Abstract

The invention discloses a Min pig whole genome liquid phase chip and application thereof. Belongs to the technical field of whole genome gene chips. The liquid phase chip comprises a probe, and a genetic typing object of the probe comprises 5K SNP (Single Nucleotide Polymorphism) loci positioned on a pig reference genome Susscrofa.Sscrofa11.1. The invention further discloses a detection method of the liquid phase chip. The Min pig genome liquid phase chip can rapidly and accurately detect the genotype of a Min pig individual, the average SNP detection rate is as high as 99.97%, and the individual detection rate is as high as 99.99%; and the genotyping stability is good, the genotype consistency can reach 99.3%, and the correlation coefficient is 99.5%. The liquid chip provided by the invention can be accurately used in Min pig character related gene positioning, Min pig genetic diversity analysis, Min pig whole genome association analysis, Min pig gene analysis and detection, and Min pig selective breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of whole genome gene chips, and more particularly to a Min pig whole genome liquid phase chip and its application. Background Art

[0002] Single nucleotide polymorphisms (SNPs) are characterized by their enormous number, dense distribution, genetic stability, ease of rapid and large-scale screening, and ease of genotyping in the genome. Hailed as a major breakthrough in 21st-century biotechnology, they possess significant biological significance. SNP genotyping technology allows for understanding genotypic differences at individual SNP sites, thereby studying the associations between genes and traits, revealing the genetic basis, and providing an important basis for disease diagnosis and drug treatment. Furthermore, in agricultural and animal husbandry breeding, SNP genotyping technology can be used to screen individuals for superior traits, accelerate breeding processes, and improve species quality and yield. With the development of high-throughput SNP genotyping technology, methods based on whole-genome or simplified genome sequencing may become the mainstream of SNP genotyping technology. For species with a reference genome sequence, SNP sites can be identified by comparing the differences between sequencing data and the reference sequence, allowing for genotyping.

[0003] Currently, commercially available pig SNP microarrays are primarily based on the Illumina and Affymetrix platforms. While Illumina chips are widely used in the market, they remain relatively expensive for practical applications in molecular breeding. They also have technical limitations, such as only being able to detect known mutations, not de novo mutations, and limited flexibility. These issues can affect the accuracy and reliability of molecular breeding experiments.

[0004] In summary, how to provide a whole-genome liquid phase chip for the Min pig population is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a Min pig whole genome liquid phase chip and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The SNP molecular marker combination used for pig genotyping consists of 5000 SNP molecular markers, which are shown in Table 1.

[0008] The above-mentioned SNP molecular marker combination is used in the preparation of a pig whole genome 5K SNP chip, or a pig genome detection reagent or kit.

[0009] A porcine whole genome 5K SNP chip comprises 5000 SNP molecular markers, as shown in Table 1.

[0010] A pig whole genome detection reagent or kit comprises 5000 SNP molecular markers, wherein the SNP molecular markers are shown in Table 1.

[0011] The application of the above-mentioned SNP molecular marker combination for pig genotyping or the above-mentioned pig whole genome 5K SNP chip or the above-mentioned pig whole genome detection reagent or kit in the positioning of genes related to Min pig traits, Min pig genetic diversity analysis, Min pig whole genome association analysis, Min pig gene analysis and detection, and Min pig selective breeding.

[0012] Application of the above-mentioned porcine whole genome 5K SNP chip in medium and high density chip filling.

[0013] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The Min pig whole genome 5K liquid phase chip provided by the present invention can quickly and accurately detect the genotype of Min pig individuals, with an average SNP detection rate of up to 99.97% and an individual detection rate of up to 99.99%. In addition, the genotyping stability is good, with a genotype consistency of up to 99.3% and a correlation coefficient of 99.5%. It can be seen that the liquid phase chip of the present invention can be accurately used for Min pig trait-related gene positioning, Min pig genetic diversity analysis, Min pig whole genome association analysis, Min pig genotyping detection, and Min pig selection breeding. The present invention not only helps to shorten the breeding cycle and improve breeding efficiency and quality, but also can detect the genotype of Min pig individuals and predict their production performance indicators such as growth rate and lean meat rate, thereby optimizing the production process and reducing breeding costs.

[0015] (2) Compared with high-density chips, the 5K liquid phase chip detection of the whole genome of Min pigs described in the present invention has lower cost and is more suitable for large-scale Min pig genotyping detection, which promotes the large-scale application of genomic selection breeding technology in pig farms, further improves the independent breeding ability of Min pigs, and helps revitalize the Min pig breeding industry.

[0016] (3) The present invention's whole-genome selective liquid phase chip for Min pigs can be supplemented with SNP sites at any time, and new probes designed based on the new sites can be added to the existing probe mixture. The present invention can also remove unnecessary SNP markers to meet the needs of different detection projects, and has high application value in the field of Min pig molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0018] Figure 1 This is a diagram showing the spacing between adjacent SNP sites in the liquid phase array of the whole genome of Min pigs in Example 1 of the present invention;

[0019] Figure 2 This is a distribution diagram of the number of SNP sites on different chromosomes in the Min pig whole genome liquid phase chip in Example 1 of the present invention, where Tilling_order1 represents VIP, QTL sites, GWAS sites, important genes, and common genes; Tilling_order2 represents annotated promoters, terminators, and variable splicing; Tilling_order3 represents the whole genome coverage sites. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0022] Example 1

[0023] Method for obtaining 5K SNP sites

[0024] S1. Use the phenotypic and genotypic data collected on site to conduct genome-wide association studies (GWAS) to obtain SNP sites related to economic traits.

[0025] A GWAS was conducted on historical data. The specific steps were as follows: First, the obtained genotypic and phenotypic data were quality controlled. The quality control criteria were as follows: individuals with missing phenotypes or phenotypic values ​​outside the range of the mean + 3 standard deviations were deleted, and the phenotypic data were tested for normality. Individuals with high genotype missing rates and data with extremely high and low individual heterozygosity (locus with a genotype missing rate >10%; loci with a minimum allele frequency less than 0.01; loci with a Hardy-Weinberg test P value <0.000001) were excluded. Genotypes were imputed for sparsely missing loci using Beagle 5.0 software. Principal component analysis and kinship analysis were performed to analyze the population structure. The top three PCA and kinship matrices with the largest explained variance were incorporated into a mixed linear model for analysis. Finally, 19 SNPs associated with the trait were identified.

[0026] S2. SNP sites obtained from pig deep resequencing detection.

[0027] To achieve comprehensive genetic coverage of the Min pig population, the inventors divided the population into families and collected 120 samples from different families. Genotyping was performed using 10x resequencing. After genome quality testing, DNA fragment amplification, fragment purification, DNA library construction, and fragment probe hybridization capture, the samples were sequenced and aligned to the porcine reference genome, Sus_scrofa.Sscrofa11.1, for variant detection. A total of 13,363,525 SNPs were identified. Using the FSFS algorithm, sites with linkage disequilibrium (r²) greater than 0.5 were clustered together, initially reducing the array density to 39,427 sites. Sites with a MAF greater than 0.2 were then screened to ensure site polymorphism, reducing the density to 15,238 sites. Finally, using the MOLO algorithm, while maintaining site polymorphism, the density was reduced to 5,560 sites.

[0028] S3. Acquisition of the Minzhu 5K SNP set.

[0029] The above two site data sets were merged as the total SNP set of the Min pig whole genome and aligned to the pig reference genome "Sus_scrofa.Sscrofa11.1" version. Duplications were removed, linkage sites were removed, and sites with many flanking interfering SNPs were removed. The obtained functional gene sites, terminators, variable splicing, and non-synonymous mutation sites on the genes were listed as important sites, and the whole genome coverage sites were listed as secondary sites. The sites were scored according to priority. According to the scoring results, a total of 5K high-quality SNP sites were obtained as the Min pig whole genome selection liquid phase chip site set of the present invention. The positions of the 5K SNP sites on the Min pig reference genome Sus_scrofa.Sscrofa11.1 are shown in Table 1.

[0030] Table 1 Collection of selected liquid phase microarray sites in the whole genome of Min pig

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050] The SNP sites of the Min pig genome-wide selection liquid phase chip obtained in this example are as follows Figure 1 、 Figure 2 As shown, Figure 1 This shows that the SNP interval of the Min pig whole genome selection liquid phase chip of the present invention is relatively reasonable. Figure 2 This indicates that the SNP markers screened by the Min pig whole genome selection liquid phase chip of the present invention cover every chromosome and are basically designed according to the chip design principles.

[0051] Example 2

[0052] Preparation of a Min pig whole genome liquid phase chip

[0053] A Minzhu whole genome liquid phase chip consists of independently packaged Minzhu 5K probe mixture and hybridization capture reagent.

[0054] The Minzhu 5K probe is a double-stranded DNA probe, based on a nucleotide sequence designed and synthesized based on the SNP site described in Example 1. The probe was designed and synthesized using targeted capture sequencing technology, based on the location of the SNP site and the sequences flanking it. The probe design principles include a probe length of 120 bp, a GC content between 30% and 80%, ≤5 regions of homology, and a biotin-modified group at the 5' end.

[0055] The hybridization capture reagent includes a biotin-labeled probe, a hybridization buffer, a blocking reagent, capture magnetic beads, and an elution buffer.

[0056] Example 3

[0057] Quality evaluation of Min pig whole-genome liquid phase array genotyping

[0058] 45 Min pigs from the experimental pig farm of the Animal Husbandry Research Institute of Heilongjiang Academy of Agricultural Sciences were randomly selected for genotyping using the Min pig whole genome liquid phase chip obtained in Example 2, and the following quality evaluation was performed.

[0059] 1. Detection rate

[0060] SNP detection rate and individual detection rate are important indicators for measuring chip quality, and are generally measured using loci on autosomes and chromosomes.

[0061] Phylogenetic identification was performed using the 5K porcine whole-genome SNP chip provided in Example 2 of the present invention. Specifically, the target individuals were genotyped using the SNP chip of the present invention. A kinship matrix was then established using the genotype information. Phylogenetic identification was performed based on the kinship coefficients between individuals combined with pedigree records (e.g., the kinship coefficient between parent and child and full siblings is approximately 0.5, and the kinship coefficient between half siblings is approximately 0.25).

[0062] The SNP detection rate of the Min pig whole genome liquid phase chip obtained in Example 2 was very high, with an average SNP detection rate of 99.97%.

[0063] The detection rates of the 45 individual SNPs were all greater than 99%, with the maximum and minimum individual detection rates being 99.99% and 99.95% respectively. The average detection rate was 99.96% with a standard deviation of 0.0055.

[0064] This shows that the quality of the Min pig whole genome liquid phase chip genotype detection of the present invention is very good.

[0065] 2. Genotyping stability

[0066] Stability is generally measured by the consistency and correlation coefficient of the genotype test results of two repeated samples.

[0067] The method for genome typing using the above-mentioned Min pig whole genome liquid phase chip comprises the following steps:

[0068] A high-throughput sequencing library of Min pig DNA is constructed using the genomic DNA of the Min pig to be tested; the above-mentioned Min pig whole-genome liquid phase chip is mixed with the constructed Min pig DNA high-throughput sequencing library to capture the target sequence hybridized with the probe; the obtained target sequence is eluted, amplified and purified, and the resulting product is subjected to high-throughput sequencing. The sequencing results are then compared to the Min pig reference genome to obtain the genomic genotyping of the Min pig to be tested.

[0069] The method for constructing a high-throughput sequencing library for Min pig DNA is as follows: the genomic DNA of the Min pig to be tested is randomly sheared using an ultrasonic disruptor, and the library is constructed through end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification steps. Preliminary quantification is performed using Qubit 2.0, the library is diluted, and then the insert fragments of the library are detected using Agilent 2100. After the insert fragment size meets the expectation, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library.

[0070] The method for capturing the target sequence hybridized with the probe is as follows: using the biotin-modified probe to hybridize with the target region of the library genome to form a double strand; then using streptavidin-coated magnetic beads to molecularly adsorb the biotin-modified probe, and then washing away the DNA fragments in the non-target region through elution treatment, thereby capturing the target sequence hybridized with the probe.

[0071] The high-throughput sequencing data were filtered using fastp software, and the SNP genotyping of individual specific sites was obtained after processing with BWA and GATK software.

[0072] Three Min pigs were used to test the Min pig whole genome liquid phase array obtained in Example 2. The results of two genotyping tests on three replicates were as follows: genotypic consistency was 99.3% and the correlation coefficient was 99.5%. This shows that the Min pig whole genome liquid phase array genotyping test described in the present invention is very stable.

[0073] From the above results, it can be seen that the use of the Min pig whole genome liquid phase chip described in the present invention can improve the accuracy of large-scale genotyping of Min pigs.

[0074] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0075] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SNP molecular marker combination for pig genotyping, characterized in that: It consists of 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

2. Use of the SNP molecular marker combination according to claim 1 in the preparation of a pig whole genome 5K SNP chip, or a pig genome detection reagent or kit.

3. A pig whole genome 5K SNP chip, characterized by: It contains 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

4. A pig whole genome detection reagent or kit, characterized in that: It contains 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.

5. Use of the SNP molecular marker combination for pig genotyping according to claim 1, the pig whole genome 5K SNP chip according to claim 3, or the pig whole genome detection reagent or kit according to claim 4 in the positioning of genes related to Min pig traits, Min pig genetic diversity analysis, Min pig whole genome association analysis, Min pig gene analysis and detection, and Min pig selective breeding.

6. Use of the porcine whole genome 5K SNP chip according to claim 3 in filling medium and high density chips.

Images