A SNP molecular marker combination applicable to Chinese indigenous pigs, a 66K SNP liquid chip, and applications thereof

Abstract

The present invention discloses a SNP molecular marker combination applicable to Chinese indigenous pigs, a 66K SNP liquid chip and applications thereof. The SNP molecular marker combination applicable to Chinese indigenous pigs consists of 66,443 SNP molecular markers, and the positions of the SNP molecular markers on the pig reference genome (Sus_scrofa.Sscrofa11.1) are shown as NO.00001 to NO.66443 in Table 1 of the specification. The SNP loci included in the liquid chip are derived from individuals of Chinese indigenous pigs, wild boars, and Western pig populations, as well as whole-genome resequencing data of 40 Beijing Black pigs, 50 Damin hybrid pigs, and 20 Jianli pigs as germplasm materials. The present invention provides an important tool for the high-precision variety identification of Chinese indigenous pigs; the chip can be widely promoted and applied in Chinese indigenous pig genetic breeding enterprises.

Description

Technical Field

[0001] The present invention relates to a SNP molecular marker combination and a liquid chip prepared therefrom, and particularly to a whole-genome liquid chip for Chinese indigenous pigs containing 66,443 SNP marker sites developed based on targeted capture high-throughput sequencing technology and its application. Background Art

[0002] With the increasing number of Chinese indigenous pig-breed new varieties containing Chinese indigenous pig bloodlines, the market share of Chinese indigenous pigs has been increasing year by year, and genomic selection breeding of Chinese indigenous pigs has become a current breeding hotspot. Low-cost, large-scale, and appropriate number of genomic loci genotyping is crucial for molecular breeding research of pigs. In recent years, the currently mainstream 50K chips (Neogen 50K or Zhongxin No. 1) based on the Illumina platform have been widely used in research such as genome-wide association analysis (GWAS) and genomic selection (GS). However, these two chips mainly target imported lean-type pigs. Although both are designed with 50K loci, when applied to Chinese indigenous pig breeds or Chinese indigenous pig-breed new varieties containing indigenous pig bloodlines, the number of quality-controlled loci drops to 20K - 30K. Therefore, it is very necessary to develop a breeding chip of about 60K for Chinese indigenous pig breeds and apply it to genome-wide association analysis (GWAS) and genomic selection (GS). Moreover, with the gradually increasing market share of Chinese indigenous pigs, its application market is broad.

[0003] High-throughput SNP chips are indispensable important tools for carrying out molecular breeding research such as high-throughput genotyping, breed identification, genome-wide association analysis, and genomic selection of animals. The breed identification and gene research of Chinese indigenous pigs mainly adopt second-generation molecular marker technologies such as SSR (simple sequence repeat) markers and AFLP (amplified fragment length polymorphism) based on electrophoresis separation and distinguished by DNA fragment length differences; there are also some studies that adopt third-generation molecular marker technologies such as KASP (competitive allele-specific polymerase chain reaction) based on single nucleotide polymorphism (SNP), but such technologies have high technical thresholds, high costs, few loci, and low breed discrimination.

[0004] SNP chips have advantages such as convenient detection, low cost, high throughput, high sensitivity, and parallel detection. Compared with solid-phase SNP chips, liquid-phase SNP chips also have advantages such as flexibility, variability, free selection of detection throughput, and no need to design solid-phase probes. Summary of the Invention

[0005] The object of the present invention is to provide a SNP molecular marker combination for Chinese indigenous pigs based on targeted sequencing; another object of the present invention is to provide a 66k SNP liquid chip suitable for Chinese indigenous pigs, that is, to provide a special liquid chip for Chinese indigenous pigs, which can evenly cover the whole genome of Chinese indigenous pigs, and has strong representativeness, high polymorphism, high specificity and strong universality. It solves the problem of the lack of large-scale molecular marker technology for existing Chinese indigenous pigs, and provides an indispensable important tool for high-throughput genotyping, breed identification, population structure research, etc. of Chinese indigenous pigs.

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

[0007] A SNP molecular marker combination suitable for Chinese indigenous pigs, which consists of 66,443 SNP molecular markers, and the positions of the SNP molecular markers on the pig reference genome (Sus_scrofa.Sscrofa11.1) are shown as NO.00001 to NO.66443 in Table 1 of the specification.

[0008] Table 1 Positions of 66,443 SNP molecular markers

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[0155] Preferably, the SNP molecular markers applicable to Chinese indigenous pigs are obtained by screening a total of 9,378,550 SNP sites, selecting a total of 95,611 sites that evenly cover 18 porcine chromosomes and 2 sex chromosomes, analyzing the upstream and downstream sequences according to the positions of the sites on the chromosomes, designing sequencing primers, and screening SNP sites that can be used for chip development.

[0156] Preferably, the SNP molecular marker sites are obtained through data analysis of the whole-genome resequencing data of individuals using 153 individuals from Chinese indigenous pigs, wild boars, and Western pig populations, as well as 40 Beijing Black pigs, 50 Damin hybrid pigs, and 20 Jianli pigs as germplasm materials.

[0157] Preferably, the method for screening population-specific SNPs for the obtained sites is as follows: a. Using 1 Mb as a window, counting the linkage of 9,378,550 SNPs; b. Counting the SNP genotype frequencies of each population, and selecting SNPs with a minor allele frequency greater than or equal to 0.05 in 80% of the populations other than wild boars as candidate SNPs; c. According to the sliding window strategy, dividing the whole-genome region into 93,786 haplotype intervals based on genotype linkage relationship (R-square <= 0.2), and screening out the SNPs with the greatest average linkage degree to other SNPs within the haplotype intervals as candidate SNPs; d. Considering that some haplotypes are relatively complex, selecting 2-5 SNPs for some haplotypes; e. Considering the SNP function, additionally adding 3,329 SNPs that meet the conditions of having a minor allele frequency greater than or equal to 0.05 in 80% of the populations other than wild boars and having been reported to be associated with traits.

[0158] A 66K SNP liquid chip applicable to Chinese indigenous pigs is prepared by combining the aforementioned 66,443 SNP molecular markers. Preferably, the 66K SNP liquid chip applicable to Chinese indigenous pigs is a liquid chip developed based on targeted sequencing genotyping technology.

[0159] Preferably, the steps for developing a 66K SNP liquid chip applicable to Chinese indigenous pigs using targeted sequencing genotyping technology include the following:

[0160] S1. According to the indicators of SNP sites, combined with the positions of the sites on the chromosome, analyze their upstream and downstream sequences and design sequencing primers. Select the upstream and downstream sequences of the SNP sites obtained, design a liquid-phase probe covering the target SNP for each site to be tested, and label it with biotin.

[0161] S2. Based on the principle of DNA base complementary pairing, hybridize the probe with the DNA sample of the genotype to be tested in solution, and form a double-strand by complementary pairing with the target region of the genome.

[0162] S3. Use streptavidin-coated magnetic beads to adsorb and enrich the DNA fragments carrying biotin, and obtain the DNA fragments containing the target SNP sites.

[0163] S4. Mix the DNA fragments obtained by capturing and enriching all sites in all strains to be tested, add recognition sequences, and through amplification, library construction and next-generation sequencing, obtain the genotyping results of each target SNP in specific individuals. The obtained probe sequences are the information of the Chinese indigenous pig liquid-phase SNP chip.

[0164] Preferably, in step S4, 132,886 probe sequences are obtained.

[0165] Application of the 66K SNP chip applicable to Chinese indigenous pigs in the in vivo detection of pig DNA.

[0166] Preferably, the applications of the chip include: breed identification and genotyping.

[0167] The Chinese indigenous pig SNP liquid-phase chip of the present invention has multiple density combinations, is economical, efficient and highly practical, and can provide important technical support for the breed identification and population structure research of Chinese indigenous pigs. The obtained 66K SNP liquid-phase chip applicable to Chinese indigenous pigs can be applied to the high-precision breed identification work of Chinese indigenous pigs. The present invention belongs to the SNP (single nucleotide polymorphism) chip for DNA detection, relates to the animal molecular markers and gene sequencing fields of life sciences, and is a Chinese indigenous pig whole-genome liquid-phase chip containing 66,443 SNP marker sites developed based on the targeted capture high-throughput sequencing technology, providing an important tool for the high-precision breed identification work of Chinese indigenous pigs; this chip can be widely promoted and applied in Chinese indigenous pig genetic breeding enterprises, and will comprehensively improve the efficiency of Chinese indigenous pig breed identification and breed protection. Description of the Drawings

[0168] Figure 1 It is the genomic distribution of the 66k SNP liquid-phase chip sites of the present invention.

[0169] Figure 2For the principle of the targeted genotyping technology of the liquid-phase probe hybridization of the present invention;

[0170] Figure 3 For the polymorphism of the 66k SNP liquid-phase chip of the present invention in 31 pig populations;

[0171] Figure 4 For the GWAS results of using this 66k SNP liquid-phase chip in Beijing Black pigs of the present invention. Detailed implementation manners

[0172] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0173] Example 1

[0174] Select the individual re-sequencing data of 153 reported pigs from Chinese indigenous pigs, wild boars, and Western pig populations, and use 40 Beijing Black pigs, 50 Damin hybrid pigs, and 20 Jianli pigs as germplasm materials for re-sequencing. Refer to Table 2 to obtain more than 9 million SNPs with a deletion of less than 10%, and SNP sites with a minor allele frequency greater than or equal to 0.05 in 60% of the populations; then, according to the indicators of these SNP sites, combined with the position of the sites on the chromosome and the linkage with other markers, as well as the minor allele frequency in each population, analyze the upstream and downstream sequences, and select 66,443 SNP sites that are highly representative, have good polymorphism, and are evenly distributed on the chromosome for chip development; finally, use the targeted sequencing genotyping (Genotyping by Targeted Sequencing, GBTS) technology, that is, a liquid-phase chip technology that only performs targeted deep re-sequencing on these 66,443 target sites, to develop a 66K SNP liquid-phase chip for Chinese indigenous pigs, which is named Zhongchu Tianpeng SNP66K chip.

[0175] Table 2 Pig breed information for liquid-phase chip development

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[0178] Example 2

[0179] A 66K SNP liquid-phase chip applicable to Chinese indigenous pigs is obtained by the following method.

[0180] (1) Re-sequencing of Chinese indigenous pigs and identification and screening of SNP sites

[0181] S1. To ensure the representativeness of Chinese indigenous pig materials, 40 Beijing Black pigs, 50 Min white pigs and Min pig crossbred pigs, and 20 Jianli pigs were used as germplasm materials. Pig blood samples were taken, and total DNA was extracted using an animal total DNA extraction kit (Tiangen, Beijing); the extracted DNA was subjected to agarose gel electrophoresis quality inspection and its concentration was measured, and then it was stored for later use at 200 ng / μl.

[0182] After the extracted DNA passed the quality inspection, it was used for high-throughput DNA sequencing; first, a DNA-seq sequencing library was constructed. The library construction was carried out using standard procedures, that is, the Illumina standard method was used for DNA library construction. For details, refer to the Illumina instruction manual (BGI provided technical services for library construction and sequencing). The DNA fragment length was 500 bp, the sequencing strategy was PE150, the sequencing depth was 30×, and 70 G of data was measured for each strain (BGI provided technical services for library construction and sequencing). Second, individual re-sequencing data based on 153 samples from Chinese indigenous pigs, wild boars, and Western pig populations were downloaded.

[0183] S2. After sequencing, the raw sequencing data obtained from the above sequencing / downloaded database was quality controlled using the sequence quality control software Trimmomatic software to remove low-quality sequences and adapter sequences, etc., that is, to remove the adapters added to both ends of the reads during the sequencing process to reduce the alignment error rate and obtain high-quality clean reads; using the pig Sscrofa11.1 (GCA_000003025.6) genomic information as the reference genome, the obtained clean reads were mapped to the reference genome using the BWA-mem2 software to obtain the alignment result files of each sample.

[0184] S3. Use the MarkDuplicates tool in the GATK software to mark the duplicate sequences in the alignment results, thereby filtering out the duplicate sequences generated by PCR to obtain a new alignment file; then use the HaplotypeCaller tool of GATK to identify genomic variation events in the obtained alignment file to obtain single nucleotide polymorphisms (SNPs) and multi-base insertion or deletion mutations (INDELs); call the VariantFiltration subroutine in the GATK software to filter all variation events to obtain a high-quality variation dataset; use the SelectVariants tool of GATK to screen out SNP loci from all variation events, and use GATK hard filtering for variant identification and screening. The screening parameters are as follows: QD < 2.0, FS > 60.0, SOR > 3.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0.; Use the PLINK software to perform the HWE equilibrium test, and filter out the variations with an HWE test p-value less than 1e-6. Subsequently, filter out the SNPs with a deletion greater than 10% and the SNP loci with a minor allele frequency greater than or equal to 0.05 in 60% of the population to obtain the final variation set, and a total of 9,378,550 SNPs are obtained.

[0185] (2) Select SNP loci for chip development

[0186] S1. Perform population-specific SNP screening on the obtained loci: a. Use 1 Mb as a window to count the linkage of 9,378,550 SNPs; b. Count the SNP genotype frequencies of each population, and select the SNPs with a minor allele frequency greater than or equal to 0.05 in other 80% of the populations except wild boars as candidate SNPs; c. According to the sliding window strategy, divide the whole genome region into 93,786 haplotype intervals according to the genotype linkage relationship (R-square <= 0.2), and select the SNP with the greatest average linkage degree with other SNPs in the haplotype interval as the candidate SNP; d. Considering that some haplotypes are relatively complex, select 2 - 5 SNPs for some haplotypes; e. Considering the SNP function, additionally add 3,329 SNPs that meet the condition of having a minor allele frequency greater than or equal to 0.05 in other 80% of the populations except wild boars and have been reported to be associated with traits.

[0187] S2. Select 95,611 loci that evenly cover 18 autosomes and 2 sex chromosomes of pigs from the SNP loci obtained in step (1).

[0188] S3. Analyze the upstream and downstream sequences of these loci according to their positions on the chromosome, determine whether it is appropriate to design probes (sequencing primers), design relevant sequencing primers, and finally screen and obtain 66,443 SNP loci that can be used for chip development, which evenly cover the whole genome, have high polymorphism, strong specificity, and good universality.

[0189] The distribution of the SNP loci screened in the above step (2) on 18 autosomes and 2 sex chromosomes of Chinese indigenous pigs is as Figure 1 shown.

[0190] (3) Develop a 66K SNP liquid chip for Chinese indigenous pigs

[0191] Develop the selected 66,443 candidate SNP loci into a 66K SNP liquid chip for Chinese indigenous pigs using the targeted sequencing genotyping technology (Genobaits technology). The targeted sequencing genotyping technology, also known as the liquid chip technology, is a technology that captures target loci and then performs deep resequencing, maps them to the reference genome, and finally obtains SNP genotyping results. This technology involves targeted capture by liquid-phase probe hybridization and high-throughput DNA sequencing technology.

[0192] Specifically, the targeted genotyping technology of liquid-phase probe hybridization is based on the principle of DNA base complementary pairing (the principle is as Figure 2 shown). Design a probe covering the target SNP (single nucleotide polymorphism) for each locus to be tested, label all probes with biotin, and the probes can form double-strands by complementary pairing with the target region of the genome in the liquid state. Use streptavidin-coated magnetic beads to adsorb the molecules carrying biotin, and finally the genotyping status of the target SNP in a specific individual can be restored through elution, amplification, library construction, and sequencing.

[0193] The specific process is as follows:

[0194] S1. Design a specific liquid-phase probe covering the target SNP for each locus to be tested according to the upstream and downstream sequences of these 66,443 candidate SNP loci, and label it with biotin.

[0195] S2. Based on the principle of DNA base complementary pairing, hybridize the probe with the DNA sample of the genotype to be tested in the solution, so as to form double-strands by complementary pairing with the target region of the genome in the liquid state.

[0196] S3. Use streptavidin-coated magnetic beads to adsorb and enrich the DNA fragments carrying biotin, and obtain DNA fragments containing the target SNP loci.

[0197] S4. Mix the DNA fragments obtained by capturing and enriching all loci in all test strains, add an identification sequence (barcode), and after amplification, library construction, and second-generation sequencing, the genotyping results of each target SNP in a specific individual can be obtained through high-throughput, achieving high-throughput SNP genotyping; that is, these 66,443 probe sequences are the information of the developed 66K liquid-phase SNP chip for Chinese indigenous pigs.

[0198] The present invention has developed a liquid-phase chip for Chinese indigenous pigs containing 66,443 SNP loci. Each locus in the chip has good specificity, strong universality among pig breeds, and high polymorphism. See Figure 3 。

[0199] Example 3

[0200] To further evaluate the value of the 66K SNP liquid-phase chip in the actual genome-wide breeding of Chinese indigenous pig breeds, it is now applied to the genome-wide association analysis and genome-wide prediction of the Chinese Min pig population. The specific process and results are as follows.

[0201] Genotype 428 Beijing Black pig individuals using the liquid-phase SNP chip of the present invention. A total of 60,607 polymorphic loci are obtained. During the association analysis and genome-wide prediction, use PLINK to calculate the kinship matrix between samples as the polygenic genetic background, use EMMAX for genome-wide association analysis, and use the R package KAML for genome-wide prediction. The results show that the 66K liquid-phase SNP chip can identify trait-associated loci. See Figure 4 The prediction accuracy for different traits ranges from 0.44 to 0.81. See Table 3.

[0202] Table 3 Genome-wide prediction accuracy of 5 traits of Beijing Black pigs using the 66k SNP chip of the present invention

[0203]

Claims

1. Application of a SNP molecular marker combination applicable to Chinese indigenous pigs in pig genotyping and breed identification, characterized in that: The SNP molecular marker combination consists of 66,443 SNP molecular markers, and the positions of the SNP molecular markers on the pig reference genome Sus_scrofa.Sscrofa11.1 are shown as NO.00001 to NO.66443 in Table 1 of the specification. The breeds include individuals of Chinese indigenous pigs, wild boars, Western pig populations, as well as Beijing Black pigs, Damin hybrid pigs, and Jianli pigs.

2. The application according to claim 1, wherein: The SNP molecular markers are obtained by screening a total of 9,378,550 SNP loci, selecting a total of 95,611 loci that evenly cover 18 pig chromosomes and 2 sex chromosomes, then analyzing the upstream and downstream sequences according to the positions of the loci on the chromosomes, designing sequencing primers, and performing population-specific SNP screening on the obtained loci to obtain SNP loci that can be used for chip development.

3. The application according to claim 2, wherein: The SNP molecular marker loci are obtained through data analysis of the whole-genome resequencing data of 153 individuals from Chinese indigenous pigs, wild boars, Western pig populations, 40 Beijing Black pigs, 50 Damin hybrid pigs, and 20 Jianli pigs used as germplasm materials.

4. The application according to claim 2, wherein: The method for performing population-specific SNP screening on the obtained loci is as follows: a. Using 1 Mb as a window, statistically analyze the linkage of 9,378,550 SNPs; b. Statistically analyze the SNP genotype frequencies of each population, and select SNPs with a minor allele frequency greater than or equal to 0.05 in 80% of the populations other than wild boars as candidate SNPs. c. According to the sliding window strategy, divide the whole-genome region into 93,786 haplotype intervals based on the genotype linkage relationship R-square <= 0.2, and select the SNP with the greatest average linkage degree with other SNPs in the haplotype interval as a candidate SNP; d. Considering that some haplotypes are relatively complex, select 2 - 5 SNPs for some haplotypes; e. Considering the SNP function, additionally add 3,329 SNPs that meet the condition of having a minor allele frequency greater than or equal to 0.05 in 80% of the populations other than wild boars and have been reported to be associated with traits.

5. A 66K SNP liquid chip applicable to Chinese indigenous pigs, characterized in that: The liquid chip described includes a set of nucleotide probes for detecting 66,443 SNP loci, and the physical positions of the 66,443 SNP loci are determined based on their positions on the pig reference genome Sus_scrofa.Sscrofa11.1; the 66,443 SNP loci are shown as NO.00001 to NO.66443 in Table 1 of the specification.

6. A design method of a 66K SNP liquid-phase chip applicable to Chinese indigenous pigs according to claim 5, characterized in that, It includes the following steps: S1 Identify and screen high-quality SNP loci in the whole genome through whole-genome resequencing of Chinese indigenous pigs. S2 Select 66,443 SNP loci for chip development. S3 Design liquid-phase probes based on the 66,443 SNP loci selected in step S2, and prepare a 66K SNP liquid chip for Chinese indigenous pigs.

7. The design method of the 66K SNP liquid-phase chip applicable to Chinese indigenous pigs according to claim 6, wherein The step S3 described includes the following steps: S31. According to the indicators of SNP loci, combined with the positions of the loci on the chromosome, analyze their upstream and downstream sequences and design sequencing primers. Select the upstream and downstream sequences of the SNP loci obtained from the SNP loci that can be used for chip development. Design a liquid-phase probe covering the target SNP for each locus to be tested, and label it with biotin; S32. Based on the principle of DNA base complementary pairing, hybridize the probe with the DNA sample of the genotype to be tested in solution, and form a double-strand by complementary pairing with the target region of the genome; S33. Use streptavidin-coated magnetic beads to adsorb and enrich the DNA fragments carrying biotin, and obtain the DNA fragments containing the target SNP loci; S34. Mix the DNA fragments obtained by capturing and enriching all loci in all strains to be tested, add an identification sequence, and through amplification, library construction and next-generation sequencing, obtain the genotyping results of each target SNP in a specific individual. The obtained probe sequences are the information of the Chinese indigenous pig liquid-phase SNP chip.

8. The design method of the 66K SNP liquid-phase chip applicable to Chinese indigenous pigs according to claim 7, characterized in that, In step S34, the number of obtained probe sequences is 132,886.

9. Application of the 66K SNP chip applicable to Chinese indigenous pigs described in claim 5 in the in vivo detection of pig DNA.

10. The application according to claim 9, characterized in that : The said application includes breed identification and genotyping. The breeds are individuals of Chinese indigenous pigs, wild boars, Western pig populations, as well as Beijing Black pigs, Damin hybrid pigs and Jianli pigs.

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