CN110191965A - Pig full-length genome 50K SNP chip and application - Google Patents
Pig full-length genome 50K SNP chip and application Download PDFInfo
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Abstract
The present invention provides a boar full-length genome 50K SNP chip and application, the SNP marker mostly come from following 3 class: the first kind, gets SNP site according to the significant site screening for having existing Important Economic functional gene reported in the literature；Second class, the good SNP site of polymorphism filtered out from domestic and international multiple kind sequencing datas；Third class derives from snp database, and total 3 class probes include 50000 SNP sites.Pig full-length genome 50K SNP chip provided by the invention can be used for specificity identification Europe pig and Asia pig, carry out full-length genome selection and use, objective trait QTL, association site and candidate gene identification, Relationship iden- tification etc..
Description
The invention relates to the technical fields of molecular biology, functional genomics, bioinformatics and genome breeding, in particular to a pig whole genome 50K SNP chip and application thereof.
SNP refers to the variation of a single nucleotide on the genome level, and comprises molecular markers formed by deletion, insertion, conversion, transversion and the like of a single base, and has the characteristics of large quantity and wide distribution. SNPs, which are genetic markers, contribute to genetic variation of complex traits, and are therefore widely used in genetic research. The SNP chip is produced by fixing a DNA probe with a fluorescent label on a silicon chip, and then performing SNP typing by hybridizing the probe DNA with genome DNA. SNPs bind to probes on the surface of the silicon wafer rather than genomic sequences, so that a large number of individual DNAs can be immobilized on one chip for analysis.
The Infinium SNP chip technology of Illumina is a relatively mature and widely applied whole-gene SNP detection platform at present. Firstly, hybridizing denatured DNA with a probe on a chip microbead, then removing DNA which is not hybridized and combined, and performing extension reaction of a special labeled base on the obtained DNA; and finally, converting SNP information into visible fluorescence information through immunological combination of the label and the fluorescent group.
At present, a second generation sequencing technology-based SNP novel high-throughput molecular marker technology has been widely applied to pigs. The most common chips in pigs at present are two 60K chips (PorcineSNP60Beadchip) from Illumina, about 6 ten thousand sites, but the distribution uniformity of the sites is general, and the positions of 10K multiple sites on chromosomes are unknown. It has been shown that the accuracy of genome selection is related to the distribution of SNP molecular markers, and the more evenly the SNP is distributed on the chromosome, the higher the accuracy of genome selection. In genome selection breeding. It is generally accepted that the higher the SNP molecular marker density, the higher the genome selection accuracy. However, the study by Su et al in 2012 showed that the accuracy was only improved by about 1% when the density of the markers was increased from 54K to 777K, since most of the markers had no effect in practical applications. Therefore, simply seeking to increase the number of markers not only increases accuracy to a limited extent but also increases cost. The invention is provided by comprehensively considering the factors.
Disclosure of Invention
The invention aims to provide a pig whole genome 50K SNP chip and application thereof.
The 50K SNP chip of the whole genome of the pig comprises SNP molecular markers shown as SEQ ID NO 1-50000.
The SNP molecular markers mainly come from 3 types of SNP loci: the first type is a remarkable locus of a gene with important economic functions according to the existing literature reports, and comprises 5788 SNP loci; the second type is SNP loci with good polymorphism screened from sequencing data of a plurality of pig breeds at home and abroad, and comprises 35765 SNP loci; the third category, which is the region not covered by the first two categories of SNPs supplemented from the NCBI porcine SNP database, contains 8447 SNP sites. The above 3 types of probes contain 50000 SNP sites in total. The chip is a fiber bead chip manufactured by Infinium patent manufacturing technology (US6,429,027) of Illumina.
The invention also provides a method for preparing the gene chip, which is used for the specific probe of the pig SNP detection chip, wherein the nucleotide sequences of the SNP markers are respectively shown as SEQ ID NO. 1-50000. The SNP locus on the pig whole genome SNP chip of the invention refers to the 71 th base of each sequence in the sequences shown in SEQ ID NO 1-50000. Then, an SNP chip is manufactured by using an Infmium chip manufacturing technology.
The invention also provides 24 SNP molecular markers for identifying the Chinese and foreign pig breeds, the SNP molecular markers are selected from at least one of the following SNP molecular markers, the nucleotide sequences are respectively shown as SEQ ID NO:1435,1895, 3713, 3716, 3717, 3719, 4298, 6716, 11665, 12437, 13507, 13540, 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078, the 71 th base of each sequence is a SNP mutation site, and the site is associated with European and Asian pig breeds.
The invention also provides application of the SNP molecular marker in pig genotyping detection alone or in combination.
The invention also provides application of the SNP molecular markers in preparation of SNP typing chips singly or in combination.
The invention also provides application of the SNP molecular marker in preparation of a pig whole genome SNP chip singly or in combination.
The invention also provides an SNP typing chip which comprises at least one of the SNP molecular markers.
The invention also provides application of the SNP molecular marker in identifying European and Asian pig breeds singly or in combination.
The invention also provides application of the SNP molecular marker in pig molecular marker assisted breeding alone or in combination.
The invention also provides application of the SNP typing chip or the 50K SNP chip in identifying European and Asian pig breeds.
The invention also provides application of the SNP typing chip or the 50K SNP chip in pig whole genome selective breeding.
The invention also provides application of the SNP typing chip or the 50K SNP chip in pig whole genome association analysis.
The invention also provides application of the SNP typing chip or the 50K SNP chip in cluster analysis and genetic relationship identification of pigs.
Compared with the prior art, the invention has the following advantages:
the SNP molecular markers for identifying Chinese and foreign pig breeds have the characteristics of uniform SNP locus distribution and high whole genome coverage rate, and the SNP markers are used for whole genome breeding, so that the association degree with characters can be effectively improved through linkage disequilibrium, and the accuracy of breeding value estimation is ensured. When the SNP loci are screened, the resequencing data of the big white pigs, the long white pigs, the Duroc and the Meishan pigs are used for screening the SNP with the common minimum allele frequency of more than 0.05 of several varieties, the selected loci have higher polymorphism, and the accuracy of the genome selective breeding value can be improved. The chip increases obvious SNP sites related to growth traits, reproduction traits, health traits and the like of pigs through functional verification, and improves the practicability of the chip. Compared with characteristic sites of representative local pig breeds in China and highly bred commercial pig breeds at home and abroad, 24 Asia specific SNP mutation sites are added, and European and Asia pigs can be specifically identified. In addition, the chip can also be used for identifying target character QTL, associated sites and candidate genes, identifying genetic relationship and the like.
FIG. 1 shows the distance (unit: kb) between SNP molecular markers adjacent to SNP used in the preparation of a swine whole genome 50K SNP chip according to the present invention.
FIG. 2 shows the distribution of SNP sites in the genome-wide level.
FIG. 3 is a Manhattan chart of the results of corrected day-old whole genome association analysis of 100kg pigs in example 3 of the present invention.
FIG. 4 shows the clustering results of the Chinese and foreign breeds in example 4 of the present invention.
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 follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 acquisition of SNP molecular markers for identifying Chinese and foreign pig species and preparation method of 50K pig whole genome SNP chip
SNP locus identification process: in order to ensure that SNP loci are uniformly distributed on the whole genome level, a chromosome is divided into 48kb windows/intervals, 1-2 SNPs in the first type of probe are preferentially selected in each interval, and if the number of the SNPs is less than 1, the SNPs in the second type of probe are screened; and selecting SNP of a third type of probe when the first type of probe and the second type of probe can not cover the window after being selected.
1. Acquisition of the first type of probe (SNP site):
and combining public databases NCBI (https:// www.ncbi.nlm.nih.gov/pubmed /), QTLdatabase (https:// www.animalgenome.org/cgi-bin/QTLdb/index /) to obtain SNP (single nucleotide polymorphism) with significant QTL (quantitative trait loci) results, and internal and upstream SNP sites, whole genome association analysis and whole genome association analysis of candidate genes related to economic traits such as pig growth traits, reproductive traits and health shapes. 31,2980 site candidate SNPs were obtained by removing duplicate sites, screening for positions in the gene structure according to MAF <0.05 and site annotation (intragenic and regulatory region preference).
The length of the whole genome of the pig is about 3Gb, and in order to ensure that the SNPs are distributed as uniformly as possible on the whole genome level, the whole genome of the pig is divided into windows every 48kb, and the starting position of the next window is the SNP position selected by the previous window. The specific method for determining SNP: 1) if more than or equal to 3 alternative SNPs exist in the window, selecting two points which enable the window to be distributed most uniformly; 2) if the number of the alternative SNPs in the window is 2 or 1, the alternative SNPs directly become the final target SNPs; 3) if the window has no alternative SNP of the first type of probe, the window is reserved and waiting for alternative SNP screening of the second type of probe. Finally, 5788 SNPs of the first type probe were determined.
2. Obtaining a second type of probe:
carrying out whole genome re-sequencing on a plurality of pig breeds including Chinese cabbage, Changbai, Duroc, Meishan, Yunnan auricle pig and Tibetan pig at home and abroad, screening SNP sites meeting the condition that MAF of each breed is greater than 0.05, and removing the weight to obtain 9,383,407 SNP sites with good polymorphism. Among them, 208,608 SNPs specific to Asian pig breeds were specifically selected.
After the first type of probe is screened, filling a second type of probe for a window without SNP, wherein the specific method comprises the following steps: 1) if the window has more than 2 alternative SNPs, selecting 1 SNP closest to the site in the window; 2) if the window has only 1 SNP, the SNP directly becomes the target SNP; 3) if the window has no alternative SNP of the second type of probe, the window is reserved and waiting for alternative SNP screening of the third type of probe. Finally, 35765 SNPs of the second type of probe were determined, including 24 SNPs specific to Asian pig breeds.
3. Obtaining probes of a third type:
probes of the third type were downloaded from the porcine SNP database at NCBI. After the second type of probe is screened, filling the second type of probe in a window without SNP, wherein the specific method comprises the following steps: 1) if the window has more than 2 alternative SNPs, selecting 1 SNP closest to the site in the window; 2) if the window has only 1 SNP, the SNP becomes the target SNP directly. The SNP of 8447 probes of the third type was finally determined.
The identified SNP sites were submitted to Illumina for scoring by the Infmium iSelect scoring System (http:// www.illumina.com /), and sites with an intragenic score <0.7 and intergenic score <0.9 were removed. And selecting the SNP site closest to the deleted unqualified SNP site for supplement, and scoring again. And finally, 50,000 SNP sites are obtained by identification and screening according to the steps. 50,000 beads (beads) were required as per the Illumina Infmium iSelect HD design requirements. All 50,000 tag SNP sites are 50,000 DNA sequences shown in SEQ ID NO 1-50000. These tag sequences were designed and manufactured into Infmium SNP chips by Illumina.
The 50K SNP chip has three advantages: one is the functional relevance with economic traits. The chip collects and determines a large number of significant sites related to growth traits, reproduction traits, health traits and the like of the pigs, so that the practicability of the chip is improved; secondly, the sites are distributed uniformly (the distance between adjacent SNPs is shown in figure 1), and the polymorphism is good. The distribution quantity of the chip sites in the whole genome is uniform (the distribution quantity of the chip sites in each chromosome is shown in table 1, and the distribution level of the SNP sites in the whole genome is shown in table 2), so that the accuracy of the breeding value estimation is ensured; and the use of resequencing data ensures that the selected sites have higher polymorphism when the sites are screened; thirdly, the specificity is possessed, characteristic sites of representative local pig breeds in China and highly bred commercial pig breeds at home and abroad are screened by comparison, and 24 specific Asia SNP sites are particularly added, and specific SNP molecular marker nucleotide sequences are respectively shown as SEQ ID NO:1435,1895, 3713, 3716, 3717, 3719, 4298, 6716, 11665, 12437, 13507, 13540, 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078, so that European and Asia pigs can be specifically identified.
TABLE 1 number of chip sites distributed on each chromosome
Example 250K application of porcine Whole genome SNP chip in genome selection Breeding-Multi-field Joint genetic evaluation
And performing multi-field combined genetic evaluation by using a 50K pig whole genome SNP chip. Large white pigs from both farms in akabane and Sichuan were examined and genetically evaluated for AGE per day (AGE)/backfat thickness (BF) and total litter size (TNB) up to 100kg of body weight using a "one-step" approach. The method comprises the following specific steps:
(1) extracting DNA of big white pigs in two pig farms to carry out SNP chip detection, and carrying out quality control on the detection result of the chip, wherein the individual CR is more than 95 percent, the MAF is more than 0.01, the Hardy-Weinberg balance test is more than 0.001, 1210 pigs and 544 pigs are respectively arranged in the two pig farms after the quality control, and 42,074 high-quality SNP loci are used for whole genome selection;
(2) model:
AGE/BF＝HYSS+Litter+animal+error
TNB＝HYS+Parity+perm+animal+error
HYSS is the herd, year, season of the individual animal when the age of the day and the back fat thickness are measured
Combined systemic environmental effects of nodes and sex of individual animals;
litter is the random effect of the nest where the animal individual is born;
HYS is the comprehensive system environmental effect of the pigsty, the year and the season of the sow during farrowing;
parity is the fetal fixed effect;
perm is a random permanent environmental effect affecting the litter size of multiple births of the same sow;
animal is random additive genetic effect of animal individual, namely breeding value;
τ＝1，w＝0.05，
ω is 1, error is a random error,
h is an individual relationship matrix, A is a molecular genetic relationship matrix constructed according to a family tree, A22Corresponding elements of individuals with genotype information in an A array, a G array is a genome relation matrix constructed based on SNP markers, G is a corrected G array,
g- β G + α, wherein α and β are obtained by solving the following equations:
where Avg is the mean, diag and offdiag represent all diagonal elements and off diagonal elements, respectively.
(3) Genetic evaluation was performed using the software PI-BLUP. The software names used are: based on a preconditioned conjugate gradient method, indirectly solving mixed model equation set software V1.0, which is abbreviated as: PI-BLUP, software work registration certificate number: 2017SRBJ 0784.
(4) Results
The accuracy of the different genetic evaluation methods is shown in table 2.
TABLE 2 accuracy comparison of genetic evaluation methods
Compared with the traditional method for estimating the breeding value by only utilizing phenotypic information, the method for selecting the genome by using the pig whole genome 50K SNP chip can effectively improve the accuracy of genetic evaluation.
Example 350K application of pig Whole genome SNP chip in Whole genome correlation analysis
The 50K pig whole genome SNP chip provided by the invention is used for carrying out genotype detection on a large white pig in a certain 1214 internal Mongolia red peak, and the genotype quality control conditions are as follows: individual CR > 95%, MAF >0.01, Hardy-Weinberg equilibrium test >0.001, yielding 1006 qualified individuals and 42185 high quality SNP sites for GWAS analysis after quality control. GWAS analysis based on general Linear model (GLM model) was performed according to corrected day age of 100kg pigs. With p < 1X 10-5 as the potential correlation level and the whole genome correlation level, the resulting Manhattan plot is shown in FIG. 3. The results show that significant association sites on chromosomes 4 and 6 may be new regulatory sites.
Example 4 clustering analysis based on 50K Whole genome SNP chip
The 50K pig whole genome SNP chip provided by the invention is used for carrying out genotype detection on DNA extracted from 35 Duroc (DL), 6 Changbai pigs, 15 big white pigs, 5 European pigs (OZ), 11 Asia pigs (YZ) and 32 Mexican pigs (MS), and MEGA software is used for carrying out cluster analysis, and the result is shown in figure 4. The results show that Duroc (DL), Changbai (CB), Dabai (DB), European pigs (OZ), Asian pigs (YZ), Meishan pigs (MS) were each pooled as a first group, duroc, Dabai, Changbai and European pigs were pooled as a second group, Meishan and Asian pigs were pooled as separate second groups, and finally European pigs were pooled with Asian pigs as a third group. The result shows that the chip provided by the invention can be well applied to Asian pigs and European pigs for genetic classification and evolutionary analysis, and the identification result is accurate and reliable.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
The pig whole genome 50K SNP chip provided by the invention can be used for specific identification of European pigs and Asian pigs, target trait QTL, association locus and candidate gene identification, genetic relationship identification and the like by carrying out whole genome selective breeding.
Claims (10)
- The pig whole genome 50K SNP chip is characterized in that the 50K SNP chip comprises SNP molecular markers shown as SEQ ID NO 1-50000.
- The 50K SNP chip of claim 1, wherein the chip is used for the selection and breeding of the whole genome of the pig.
- The 50K SNP chip of claim 1, wherein the chip is used for pig genome-wide association analysis.
- The 50K SNP chip of claim 1, wherein the chip is used for cluster analysis and genetic relationship identification of pigs.
- The use of the 50K SNP chip of claim 1 for identifying European and Asian pig breeds.
- The SNP molecular marker for identifying the foreign pig breeds is characterized by being selected from at least one of the following SNP molecular markers, the nucleotide sequences are respectively shown as SEQ ID NO:1435,1895, 3713, 3716, 3717, 3719, 4298, 6716, 11665, 12437, 13507, 13540, 13586, 28056, 35883, 35970, 36063, 40325, 40889, 41835, 42501, 48782, 48813 or 49078, the 71 th base of each sequence is a SNP mutation site, and the site is associated with European and Asian pig breeds.
- Use of the SNP molecular markers according to claim 1 or 6, alone or in combination, for the genotyping detection of swine.
- The use of the SNP molecular markers according to claim 1 or 6, alone or in combination, for preparing a porcine whole genome SNP chip.
- Use of the SNP molecular markers according to claim 6, alone or in combination, for identifying European and Asian pig breeds.
- The use of the SNP molecular markers according to claim 1 or 6, alone or in combination, for molecular marker assisted breeding in swine.
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| WO2019113818A1 (en) | 2019-06-20 |
| CN110191965B (en) | 2022-05-27 |
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