A SNP molecular marker combination and 50K SNP chip based on targeted capture sequencing for Jiangquhai pig and its application

Abstract

The present invention discloses a Jiangquhai pig SNP molecular marker combination based on targeted capture sequencing, a 50KSNP chip and an application thereof, wherein the Jiangquhai pig SNP molecular marker combination consists of 50014 SNP molecular markers, and the positions of the SNP molecular markers on the pig reference genome (Sus_scrofa.Sscrofa11.1) are shown in Table 1 of the specification as NO.00001 to NO.50014. The SNP sites contained in the chip are derived from the whole genome resequencing data of the Jiangquhai pig, so it is more suitable for the variety identification and kinship identification of the Jiangquhai pig; the chip is a liquid phase chip, and compared with the solid phase chip, the liquid phase chip has flexible marker addition and subtraction, flexible sample detection, and good universality of the sequencing platform.

Description

Technical Field

[0001] The invention belongs to the technical field of gene chips and relates to a Jiangquhai pig 50K SNP liquid phase chip based on targeted capture sequencing and an application thereof. Background Art

[0002] Jiangquhai pigs are gradually formed by hybridization and long-term breeding of large-faced pigs and Huai pigs, and have the characteristics of both. As early as 1818, there was a record in the book "Haiqu Shiyi" written by Jin Bang that "(local pigs) are not like the bitter-faced pigs in Huaibei", indicating that a new type different from the Huai pig had been formed at that time, namely the modern Jiangquhai pig. Jiangquhai pigs have the characteristics of docile temperament, strong adaptability, early sexual maturity, high fertility, tender meat and delicious meat. But they also have the weaknesses of slow growth and low lean meat rate. The third national livestock and poultry genetic resources census in 2021 showed that there were only 1,521 Jiangquhai pigs in Jiangsu Province, including 15 boars and 343 basic sows. The endangered level of Jiangquhai pigs is endangered, and the small conservation population is prone to increase inbreeding coefficients and thus performance decline. Therefore, breed identification and kinship identification are of great significance to the protection of Jiangquhai pigs.

[0003] Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. SNPs are numerous and widely distributed, suitable for rapid and large-scale screening, and easy to genotype. They have become an important tool for livestock and poultry breed identification and kinship identification. The current mainstream commercial pig SNP chips are solid-phase chips based on the Illumina platform, such as Neogene 50K and SMIC-1, and most of the chip sites are derived from the Duroc pig reference genome, which cannot accurately identify the breed and kinship of local pigs. Liquid-phase chips based on targeted capture sequencing technology (Genotype by Target Sequencing, GBTS) not only have good universality for sequencing platforms, but also have the advantages of flexible marker addition and subtraction and flexible sample detection, and have gradually become a revolutionary technology for SNP chips. Summary of the invention

[0004] The purpose of the present invention is to provide a Jiangquhai pig SNP molecular marker combination based on targeted capture sequencing and a 50KSNP liquid phase chip and application to achieve the variety identification and kinship identification of the Jiangquhai pig.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A Jiangquhai pig SNP molecular marker combination based on targeted capture sequencing, the Jiangquhai pig SNP molecular marker combination consists of 50014 SNP molecular markers, and the positions of the SNP molecular markers on the pig reference genome (Sus_scrofa.Sscrofa11.1) are shown in NO.00001 to NO.50014 in Table 1 of the specification.

[0007] Table 1 Positions of 50014 SNP molecular markers

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[0130] The SNP molecular markers include two categories: one category is 11,169 variety identification sites; the second category is 38,845 SNP sites screened based on the uniform distribution of the genome, namely background sites.

[0131] The breed identification loci are obtained through data analysis based on the whole genome resequencing data of Jiangquhai pigs and other pig breeds.

[0132] The background site is obtained by the following method:

[0133] The whole genome was divided into windows of every 20K, 11169 variety identification sites were used as VIP sites, and 17,403,501 sites were used as candidate sites for chip site selection; the principle of site selection in each window was: if there was a VIP site in the window, only the VIP site was retained; if there was no VIP site in the window, the site with the highest MAF in the window was selected and quality controlled according to the standards of MAF < 0.05 and Hardy-Weinberg Equilibrium (HWE) < ​​10e-6, and so on; the candidate sites were submitted to the probe design system of Beijing Compson Agricultural Technology Co., Ltd. for scoring, and finally a total of 38845 SNP sites were obtained.

[0134] A Jiangquhai pig 50K SNP chip is prepared by the Jiangquhai pig SNP molecular marker combination.

[0135] The chip is a liquid phase chip based on targeted capture sequencing technology.

[0136] The application of the Jiangquhai pig 50K SNP chip in pig DNA sample detection.

[0137] The application includes the species identification and kinship identification of Jiangquhai pig.

[0138] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0139] (1) The SNP loci contained in this chip are derived from the whole genome resequencing data of Jiangquhai pig, so it is more suitable for the species identification and kinship identification of Jiangquhai pig;

[0140] (2) This chip is a liquid phase chip. Compared with solid phase chips, this liquid phase chip has flexible labeling and sample detection, and the sequencing platform has good universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 This is the genome distribution map of the Jiangquhai pig 50K SNP liquid phase chip in Example 1 of the present invention;

[0142] Figure 2This is a PCA clustering result diagram of seven pig breeds using liquid phase chip in Example 3 of the present invention;

[0143] Figure 3 This is a cluster diagram of the kinship of 103 Jiangquhai pigs using liquid phase chip in Example 4 of the present invention. DETAILED DESCRIPTION

[0144] The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

[0145] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention.

[0146] Example 1 Design and preparation of Jiangquhai pig 50K SNP liquid phase chip

[0147] 1. Establishment of SNP background database of Jiangquhai pig

[0148] The present invention utilizes the whole genome resequencing data of 62 Jiangquhai pigs and obtains highly reliable SNP sites for subsequent screening through data analysis.

[0149] 62 Jiangquhai pigs were obtained from Jiangsu Jiangquhai Pig Seed Co., Ltd. and were sequenced using the Illumina sequencing platform with a high-throughput sequencing read length of PE150 and a sequencing depth of 10×. After the sequencing data was downloaded, FastQC was used to remove adapter-contaminated reads, low-quality reads, and reads with a N ratio greater than 5% to obtain high-quality clean reads. CleanReads were aligned to the reference genome (Sscrofa11.1) using BWA software, and mutation detection was performed using GATK software and filtered according to the following criteria: (1) depth-based variant confidence (QD) < 2.0; (2) p-value (FS) using Fisher's exact test > 60.0; (3) RMS mapping quality (MQ) < 40.0; (4) Z-score of the Wilcoxon rank sum test of Alt vs. Ref read MQs (MQRankSum) < -12.5; (5) Z-score of the Wilcoxon rank sum test of the position deviation of Alt vs. Ref reads (ReadPosRankSum) < -8.0; (6) strand bias estimated by the symmetric odds ratio test (SOR) > 3.0; (7) call rates < 0.9; (8) minor allele frequencies (MAF) < 0.01. After filtering according to this standard and removing insertion and deletion sites, a total of 17,419,845 high-quality mutation sites were obtained.

[0150] 2. Screening of variety identification sites

[0151] The present invention uses the whole genome resequencing data of 62 Jiangquhai pigs and 707 individuals of other 41 pig breeds to obtain the Jiangquhai pig breed identification site through data analysis. The whole genome resequencing data of 707 individuals of other 41 pig breeds refer to the Jiangquhai pig high-quality mutation site identification process. The MAF values ​​of 17,419,845 SNPs were calculated in 42 pig breeds respectively, and the sites with the absolute value of the difference between the MAF value of Jiangquhai pig and the maximum MAF value in other 41 pig breeds greater than 0.7 were selected as the Jiangquhai pig breed identification sites, a total of 15,984 SNPs, which were submitted to the probe design system of Beijing Compson Agricultural Technology Co., Ltd. for scoring, and finally a total of 11,169 breed identification sites were obtained.

[0152] 3. Screening of background sites

[0153] According to the principle of uniform distribution of the genome, background SNP sites were added to the chip. The whole genome was divided into a window of 20K each, 11,169 variety identification sites were used as VIP sites, and 17,403,501 sites were used as candidate sites for chip selection. The principle of selecting sites in each window is: if there is a VIP site in the window, only the VIP site is retained; if there is no VIP site in the window, the site with the highest MAF in the window is selected and quality controlled according to the standards of MAF < 0.05 and Hardy-Weinberg Equilibrium (HWE) < ​​10e-6, and so on. The candidate sites were submitted to the probe design system of Beijing Compson Agricultural Technology Co., Ltd. for scoring, and finally a total of 38,845 background sites were obtained. According to the positions and flanking sequence information of 50,014 SNP sites of 11,169 variety identification sites and 38,845 background sites, probes were designed and synthesized by Beijing Compson Agricultural Technology Co., Ltd. using targeted capture sequencing technology to prepare the Jiangquhai pig 50K SNP liquid phase chip.

[0154] Example 2 Application of Jiangquhai pig 50K SNP liquid phase chip in pig DNA sample detection

[0155] 1. Extraction of pig genomic DNA samples: Collect pig ear tissue samples and use a special kit to extract ear tissue genomic DNA.

[0156] 2. DNA sample quality detection: Nanodrop detects DNA purity, Qubit accurately quantifies DNA concentration, and agarose gel electrophoresis analysis detects DNA integrity.

[0157] 3. Liquid phase chip detection: follow the standard process of liquid phase chip detection.

[0158] 4. Data analysis: The raw data was subjected to quality control steps such as removal of connectors and filtering of low-quality data to obtain clean data. The clean data was aligned to the porcine reference genome (Sus_scrofa.Sscrofa11.1) using bwa software, and the standard process of GATK software was used to detect SNPs and perform genotyping.

[0159] Example 3 Application of Jiangquhai pig 50K SNP liquid phase chip in Jiangquhai pig breed identification

[0160] The present invention uses the Jiangquhai pig 50K SNP liquid phase chip prepared in Example 1 to perform genotyping on 151 individuals of 7 pig breeds (Table 2) (see Example 2 for the specific operation method). After quality control, sites with MAF < 0.01 and detection rate < 0.9 are removed, and 46153 SNPs remain. Principal Component Analysis (PCA) is performed on the genotype data of 151 individuals of 7 pig breeds using GCTA (V1.94) software. The PCA results are shown in Figure 2 The results showed that the Jiangquhai pig could be well distinguished from other pig species using this chip.

[0161] Table 2 Pig breeds and quantities used for breed identification

[0162] Serial number variety quantity source 1 Sea Pig with Ginger Sauce 31 Jiangsu Jiangquhai Pig Seed Co., Ltd. 2 Two-faced pig 20 Changshu Animal Husbandry and Industry Co., Ltd. 3 Fengjing Pig 13 Zhenjiang Muyuan Animal Science and Technology Development Co., Ltd. 4 Sujiang Pig 27 Jiangsu Sujiang Breeding Pig Co., Ltd. 5 Duroc pig 20 Fujian Yongcheng Agriculture and Animal Husbandry Technology Group Co., Ltd. 6 Large White Pig 20 Fujian Yongcheng Agriculture and Animal Husbandry Technology Group Co., Ltd. 7 Landrace pig 20 Fujian Yongcheng Agriculture and Animal Husbandry Technology Group Co., Ltd.

[0163] Example 4 Application of Jiangquhai pig 50K SNP liquid phase chip in identification of Jiangquhai pig kinship

[0164] This example is aimed at 103 Jiangquhai pigs from Jiangsu Jiangquhai Pig Seed Co., Ltd., including 13 boars and 90 sows. The Jiangquhai pig 50K SNP liquid phase chip prepared in Example 1 was used to perform kinship analysis and construct a molecular pedigree. Through genomic kinship analysis and cluster analysis based on the G matrix, it was found that the 103 Jiangquhai pigs can be divided into 6 families ( Figure 3 ), and according to the relationship between sows and boars, the sows were divided into different families. The inbreeding coefficient of all Jiangquhai pigs based on ROH was also calculated. The average inbreeding coefficient of the group was 0.137, and individuals with a high degree of inbreeding were avoided in subsequent breeding. In order to reduce the inbreeding increment of the group, it is not recommended to breed male and female pigs of the same family. In the subsequent breeding, the kinship coefficient was confirmed before breeding. The kinship coefficients of the six sows in the "other" group in the group family construction results with all boars were less than 0.1, and they could be bred with any boar.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Jiangqu Haizhu 50K SNP chip, characterized in that: The Jiangquhai pig 50K SNP chip includes probes for detecting the Jiangquhai pig SNP molecular marker combination, and the Jiangquhai pig SNP molecular marker combination consists of 50014 SNP molecular markers. The positions of the SNP molecular markers on the pig reference genome (Sus_scrofa.Sscrofa11.1) are shown in NO.00001 to NO.50014 in Table 1 of the specification.

2. The Jiangquhai pig 50K SNP chip according to claim 1, characterized in that: The chip is a liquid phase chip based on targeted capture sequencing technology.

3. Use of the Jiangquhai pig 50K SNP chip according to claim 1 or 2 in the detection of pig DNA samples.

4. The use according to claim 3, characterized in that: The application includes the species identification and kinship identification of Jiangquhai pig.

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