WO2019113818A1

WO2019113818A1 - Whole swine genome 50k snp chip and application thereof

Info

Publication number: WO2019113818A1
Application number: PCT/CN2017/115856
Authority: WO
Inventors: 刘剑锋; 冯文; 杜恒; 赵鹏举
Original assignee: 中国农业大学
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2019-06-20
Also published as: CN110191965A; CN110191965B

Abstract

Provided are a whole swine genome 50K single-nucleotide polymorphism (SNP) chip and an application thereof, wherein SNP molecular markers are mainly derived from the following three categories: in the first category, SNP sites are obtained by screening significant sites of existing economically important functional genes reported in the literature; in the second category, SNP sites having good polymorphism are screened from sequencing data of multiple varieties domestically and abroad; in the third category, SNP sites are derived from an SNP database; the three categories of probes comprise a total of 50,000 SNP sites. The whole swine genome 50K SNP chip may be used to specifically identify European pigs and Asian pigs for breeding on the basis of whole genome selection, identification of target trait quantitative trait loci (QTL), association sites and candidate genes, kinship identification, and so on.

Description

Pig Whole Genome 50K SNP Chip and Its Application

Technical field

The invention relates to the fields of molecular biology, functional genomics, bioinformatics, genome breeding, and in particular to a pig whole genome 50K SNP chip and application.

Background technique

SNP refers to the variation of a single nucleotide at the genomic level, including molecular markers formed by deletion, insertion, transformation and transversion of a single base, and has a large number of distributions. As a genetic marker, SNP contributes to the genetic variation of complex traits and is therefore widely used in genetic research. The SNP chip is prepared by immobilizing a fluorescently labeled DNA probe on a silicon wafer, and then performing SNP typing by hybridizing the probe DNA with the genomic DNA. The SNP binds to the probe on the surface of the wafer rather than the genomic sequence, so that a large number of individual DNA can be immobilized on a single chip for analysis.

Illumina's Infinium SNP chip technology is a mature and widely used whole-genome SNP detection platform. First, the denatured DNA is hybridized with the probe on the microbead, then the unhybridized bound DNA is removed, and the obtained DNA is subjected to a specific labeled base extension reaction; finally, the SNP is bound by immunological binding of the label and the fluorescent group. Information is transformed into visible fluorescent information.

At present, SNP new high-throughput molecular marker technology based on second-generation sequencing technology has been widely used in pigs. The most common chip currently on pigs is the two 60K chips (PorcineSNP60BeadChip) from Illumina, which has about 60,000 sites, but the distribution uniformity of these sites is general, and there are 10K sites on the chromosome. Not available. Studies have shown that the accuracy of genome selection is related to the distribution of SNP molecular markers. The more uniform the distribution of SNPs on chromosomes, the higher the accuracy of genome selection. Genomic selection in breeding. It is generally believed that the higher the SNP molecular marker density, the higher the accuracy of genome selection. However, research by Su et al. in 2012 showed that when the mark density increased from 54K to 777K, Its accuracy has only increased by about 1%, because in practice, most of the markup has no effect. Therefore, simply pursuing the increase in the number of marks not only increases the accuracy but also increases the cost. The present invention has been made in consideration of the above factors.

Summary of the invention

The object of the present invention is to provide a pig whole genome 50K SNP chip and application.

The pig whole genome 50K SNP chip provided by the present invention, the 50K SNP chip comprising the SNP molecular marker as set forth in SEQ ID NO: 1-50000.

The SNP molecular markers are mainly derived from three types of SNP loci: the first type is a significant locus with important economic functional genes reported in the literature, including 5788 SNP loci; the second type is from domestic and foreign. Sequencing data from multiple swine cultivars screened for a well-characterized SNP locus containing 35,765 SNP loci. The third category was a region that was not covered by the first two types of SNPs supplemented by NCBI's porcine SNP database. 8447 SNP sites. The above three types of probes contain a total of 50,000 SNP sites. This chip is a fiber optic microbead chip made by Illumina's patented Infinium manufacturing technology (US6,429,027).

The present invention also provides a method for preparing the above gene chip for use in a specific probe of the porcine SNP detecting chip of the present invention, wherein the SNP marker has a nucleotide sequence as shown in SEQ ID NO: 1-50000, respectively. The SNP site on the pig whole genome SNP chip of the present invention refers to the 71st base of each sequence in the sequence shown by SEQ ID NO: 1-50000. The SNP chip is then fabricated using Infmium chip fabrication technology.

The invention also provides 24 SNP molecular markers for identifying Chinese and foreign pig breeds, the SNP molecular markers being selected from at least one of the following, the nucleotide sequences are SEQ ID NO: 1435, 1895, 3713, 3716, 3717, 3719, respectively. , 7298, 6716, 11665, 12437, 13507, 13540, 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078, the 71st base of each sequence is a SNP mutation Site, which is associated with European pig and Asian pig breeds.

The invention also provides the use of the SNP molecular markers alone or in combination in the detection of porcine genotyping.

The invention also provides the use of the SNP molecular markers, alone or in combination, in the preparation of SNP typing chips.

The invention also provides for the use of the SNP molecular markers, alone or in combination, in the preparation of a pig whole genome SNP chip.

The present invention also provides a SNP typing chip, the SNP typing chip comprising at least one of the above SNP molecular markers.

The invention also provides for the use of the SNP molecular markers, alone or in combination, in the identification of European and Asian pig breeds.

The invention also provides the use of the SNP molecular markers alone or in combination in porcine molecular marker-assisted breeding.

The invention also provides the use of the SNP typing chip or the 50K SNP chip for identifying European pigs and Asian pig breeds.

The invention also provides the use of the SNP typing chip or the 50K SNP chip in pig whole genome selective breeding.

The invention also provides the use of the SNP typing chip or the 50K SNP chip in pig genome-wide association analysis.

The invention also provides the application of the SNP typing chip or the 50K SNP chip in pig cluster analysis and kinship identification.

Compared with the prior art, the present invention has the following advantages:

The SNP molecular marker provided by the invention for identifying Chinese and foreign pig breeds has the characteristics of uniform distribution of SNP sites and high coverage of whole genomes, and the whole genome breeding by using these SNP markers can effectively improve the association with traits through linkage disequilibrium. The degree ensures the accuracy of the estimation of breeding values. When SNP loci were screened, the resequencing data of Large White, Landrace, Duroc and Meishan pigs were used to screen out SNPs with several common allele frequencies >0.05, and the selected loci had higher polymorphism. It also increases the accuracy of genomic selection breeding values. This chip adds significant SNP sites related to pig growth traits, reproductive traits and health traits through functional verification, improving the usability of the chip. Comparative screening The representative local pig breeds in China and the highly selective breeding of commercial pig breeds at home and abroad have added 24 Asian-specific SNP mutation sites to specifically identify European and Asian pigs. In addition, the chip can also be used for target trait QTL, related sites and candidate gene identification, kinship identification and so on.

DRAWINGS

Figure 1 is the distance (unit: kb) of SNP molecular marker adjacent SNPs used in the preparation of a pig whole genome 50K SNP chip of the present invention.

Figure 2 is a distribution of SNP loci at the whole genome level of the present invention.

Fig. 3 is a Manhattan view showing the result of genome-wide association analysis of 100 kg corrected day old pigs in Example 3 of the present invention.

4 is a clustering result of Chinese and foreign pig breeds in Example 4 of the present invention.

Detailed ways

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are based on routine experimental conditions, such as the Sambrook J&Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or as suggested by the manufacturer's instructions.

Example 1 Acquisition of SNP molecular markers for identifying Chinese and foreign pig breeds and preparation method of 50K pig whole genome SNP chip

SNP site identification process: In order to make the SNP site evenly distributed at the genome-wide level, the chromosome is divided into 48kb a window/interval, and each interval preferentially selects 1-2 SNPs in the first type of probe, if less than 1 , the SNP in a second type of probe is screened; after the first type of probe and the second type of probe are selected, the SNP of the third type of probe is selected.

1. Acquisition of the first type of probe (SNP site):

Combine with the public database NCBI (https://www.ncbi.nlm.nih.gov/pubmed/), QTLdatabase (https://www.animalgenome.org/cgi-bin/QTLdb/index/) to obtain growth traits, Internal traits related to economic traits such as reproductive traits and healthy shapes And SNPs in the upstream and downstream SNPs, genome-wide association analysis and QTL results were significant SNPs. 31,2980 locus alternative SNPs were screened by removal of the repetitive sites, MAF < 0.05 according to the locus, and position in the gene structure (intragenic and regulatory region preferential).

The whole genome length of pigs is about 3Gb. In order to ensure that these SNPs are distributed as evenly as possible at the genome-wide level, the full length of the pig genome is divided into a window every 48kb, and the starting position of the next window is selected in the previous window. SNP location. The method for specifically determining the SNP: 1) if there are more than three alternative SNPs in the window, select two points in which the distribution of the window is the most uniform; 2) if the number of alternative SNPs in the window is 2 or 1 then the alternative SNP becomes the final target SNP; 3) if the window does not have an alternative SNP for the first type of probe, the window is retained, waiting for alternative SNP screening of the second type of probe. The SNPs of 5788 first-class probes were finally determined.

2. Acquisition of the second type of probe:

Whole genome re-sequencing of a variety of pig breeds including Dabai, Changbai, Duroc, Meishan, Minnan small-ear pigs and Tibetan pigs at home and abroad, screening SNP loci that meet the MAF>0.05 of each variety, de-weighting, 9,383,407 polymorphic SNP loci were obtained. Among them, the specific SNPs unique to 208,608 Asian pig breeds were selected.

After the first type of probe screening is completed, for the window without SNP, the second type of probe is filled, and the specific method is as follows: 1) If the window has more than 2 alternative SNPs, select the distance from the window. The most recent SNP; 2) if the window has only 1 SNP, the SNP becomes the target SNP directly; 3) If the window has no alternative SNP of the second type of probe, the window is retained, waiting for the third type of probe Alternative SNP screening for needles. The SNPs of 35,765 second-type probes were finalized, including SNPs specific to 24 Asian pig breeds.

3. Acquisition of the third type of probe:

A third type of probe was downloaded from the pig SNP database database on NCBI. After screening the second type of probe screening, for the window without SNP, the second type of probe is filled, the specific method is as follows: 1) If the window has more than 2 alternative SNPs, select the distance from the window. Point to the nearest SNP; 2) If the window has only 1 SNP, the SNP is directly For the target SNP. The SNP of the 8447 third-type probes was finally determined.

The identified SNP locus was assigned to Illumina for scoring by the Infmium iSelect scoring system (http://www.illumina.com/), and the intragenomic score <0.7 and the intergenic <0.9 loci were removed. For the deleted unqualified SNP locus, the nearest SNP locus is selected for replenishment and scored again. Following the above steps identified and screened, a total of 50,000 tag SNP sites were obtained. According to the Illumina Infmium iSelect HD design requirements, 50,000 beads are required. All 50,000 tag SNP sites are 50,000 DNA sequences as set forth in SEQ ID NO: 1-50000. These tag sequences were handed over to the Infmium SNP chip by Illumina.

The 50K SNP chip has three advantages: one is the functional relevance of economic traits. The chip collects and identifies a large number of significant sites related to pig growth traits, reproductive traits and health traits, increasing the practicality of the chip; second, the site distribution is uniform (distance of adjacent SNPs is shown in Figure 1), polymorphism it is good. The sites of the chip are evenly distributed throughout the genome (see Table 1 for the number of chromosome sites distributed in each chromosome, and the level of SNP locus in the whole genome distribution is shown in Figure 2), which ensures the accuracy of the estimation of breeding values; The use of resequencing data ensures that the selected locus has a high polymorphism; the third is specific, and compares and screens the representative local pig breeds in China, and the characteristic loci of highly selected commercial pig breeds at home and abroad, especially 24 Asian-specific SNP sites, specifically the SNP molecular marker nucleotide sequences are SEQ ID NO: 1435, 1895, 3713, 3716, 3717, 3719, 4298, 6716, 11665, 12437, 13507, 13540, respectively. European and Asian pigs can be specifically identified as indicated by 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078.

Table 1 The number of chip sites distributed in each chromosome

Example 2 Application of 50K Pig Whole Genome SNP Chip in Genome Selective Breeding - Multi-field Combined Genetic Evaluation

Multiple field combined genetic evaluations were performed using a 50K pig genome-wide SNP chip. Large white pigs from two farms in Chifeng and Sichuan were tested for genetic evaluation of 100 kg body weight (AGE)/backfat thickness (BF) and total litter size (TNB) using a “one-step method”. Specific steps are as follows:

(1) Extract the DNA of two pig farms and white pigs for SNP chip test, and carry out quality control on the chip test results, individual CR>95%, MAF>0.01, Hardy-Weinberg balance test>0.001, two pigs after quality control There were 1210 heads and 544 pigs, respectively, and 42,074 high quality SNP loci for genome-wide selection;

(2) Model:

AGE/BF=HYSS+Litter+animal+error

TNB=HYS+Parity+perm+animal+error

HYSS is the pig, year and season where the individual animal is measured when the age and back is thick.

Combined system environmental effects of the section and the gender of the individual animal;

Litter is the random effect of the nest where the animal is born;

HYS is the integrated system environmental effect of the herd, annual and seasonal season in which the sow is born;

Parity is a fixed effect of parity;

Perm is a random permanent environmental effect that affects the litter size of multiple births of the same sow;

Animal is a random additive genetic effect of an individual animal, ie breeding value;

τ=1, w=0.05,

Ω=1, error is a random error,

H is the individual relationship matrix, A matrix is the molecular affinity matrix constructed according to the pedigree, A ₂₂ is the corresponding element in the A matrix with the genotype information, and G array is the genomic relationship matrix constructed based on the SNP marker, G* is Corrected G array,

G*=βG+α, where α and β are obtained by solving the following equation:

Where Avg refers to the mean, diag and offdiag represent all diagonal elements and off-diagonal elements, respectively.

(3) Genetic evaluation using software PI-BLUP. The software name used is: indirect solution of mixed model equations software V1.0 based on preconditioned conjugate gradient method, referred to as: PI-BLUP, software book registration certificate number: 2017SRBJ0784.

(4) Results

The accuracy of different genetic assessment methods is shown in Table 2.

Table 2 Comparison of the accuracy of genetic evaluation methods

It can be seen that using the pig whole genome 50K SNP chip of the present invention for genome selection can improve the accuracy of genetic evaluation by using only phenotypic information for breeding value estimation compared with the conventional method.

Example 3 Application of 50K pig whole genome SNP chip in genome-wide association analysis

The 50K pig whole genome SNP chip provided by the present invention was used for genotyping detection of 1214 large white pigs in Chifeng, Inner Mongolia. The genotype quality control conditions were: individual CR>95%, MAF>0.01, Hardy-Weinberg equilibrium test>0.001 After quality control, 1006 eligible individuals and 42185 high quality SNP loci were obtained for GWAS analysis. A GWAS analysis based on a general linear model (GLM model) was performed according to the corrected age of pigs of 100 kg. Taking p<1×10-5 as the potential correlation level and the genome-wide correlation level, the results are shown in Figure 3. The results show that significant association sites on chromosomes 4 and 6 may be new regulatory sites.

Example 4 Cluster Analysis Based on 50K Whole Genome SNP Chip

Using the 50K pig genome-wide SNP chip provided by the present invention, 35 Duruk (DL), 6 Landrace pigs, 15 Large White pigs, 5 European domestic pigs (OZ), 11 Asian domestic pigs (YZ), and 32 heads The DNA extracted from Chinese Meishan pig (MS) was genotyped and clustered using MEGA software. The results are shown in Figure 4. The results showed that Duroc (DL), Changbai (CB), Dabai (DB), European domestic pig (OZ), Asian domestic pig (YZ), Meishan pig (MS) first gathered into the first category, Duroc, Dabai , Changbai and European domestic pigs converge into the second category, Meishan and Asian pigs gather in a separate second category, and finally European pigs and Asian pigs converge into the third category. The results show that the chip provided by the invention can be well applied to the genetic classification and evolution analysis of Asian pigs and European pigs, and the identification results are accurate and reliable.

Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Industrial applicability

The whole genome 50K SNP chip provided by the invention can be used for specific identification of European pigs and Asian pigs, target trait QTLs, related sites and candidate gene identification, and genetic relationship identification.

Claims

A pig whole genome 50K SNP chip characterized in that the 50K SNP chip comprises a SNP molecular marker as set forth in SEQ ID NOs: 1-50000.
Use of the 50K SNP chip of claim 1 for pig whole genome selection breeding.
Use of the 50K SNP chip of claim 1 in pig genome-wide association analysis.
The use of the 50K SNP chip of claim 1 for cluster analysis and phylogenetic identification of pigs.
Use of the 50K SNP chip of claim 1 for the identification of European and Asian pig breeds.
A SNP molecular marker for identifying a Chinese and foreign pig breed, characterized in that the SNP molecular marker is at least one selected from the group consisting of SEQ ID NO: 1435, 1895, 3713, 3716, 3717, 3719, 4298, respectively. , 7116, 11665, 12437, 13507, 13540, 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078, the 71st base of each sequence is a SNP mutation site This site is associated with European pig and Asian pig breeds.
The use of the SNP molecular markers of claim 1 or 6 alone or in combination for the detection of porcine genotyping.
Use of the SNP molecular markers of claim 1 or 6 alone or in combination for the preparation of a pig whole genome SNP chip.
Use of the SNP molecular markers of claim 6 alone or in combination for identifying European pigs and Asian pig breeds.
Use of the SNP molecular marker according to claim 1 or 6 alone or in combination in porcine molecular marker-assisted breeding.