CN114717329B - SNP molecular marker related to sow stillbirth traits and application thereof - Google Patents
SNP molecular marker related to sow stillbirth traits and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pig molecular markers, and in particular relates to a SNP molecular marker related to the characteristics of the stillbirth of a sowAnd recording and application thereof. The invention performs genotyping by utilizing a gene chip technology, performs whole genome association analysis based on SNP molecular markers, and screens out 6 SNP molecular marker loci related to the stillbirth traits of the large white sow. The SNP molecular marker loci are respectively positioned on the 15 th chromosome of the sowSLC4A10A gene fragment. The invention provides new SNP molecular marker resources for the sow stillbirth traits, and can be applied to marker-assisted selection of sow genetic breeding.
Description
Technical Field
The invention belongs to the technical field of pig molecular markers, and particularly relates to an SNP molecular marker related to the characteristics of a sow stillbirth and a fetus and application thereof.
Background
In China, pork yield and sales volume are in the front of the world, and the stock quantity of live pigs and stock quantity of sows which can be bred are obviously reduced due to the influence of epidemic diseases, so that the improvement of the reproductive performance of the sows is particularly important under the situation. Reproductive traits are one of important economic traits of sows, and molecular breeding is performed aiming at molecular marker loci influencing target traits, so that breeding efficiency and reproductive performance of the sows can be effectively improved (Wang Yuan, 2018). In recent years, high-throughput sequencing technology and gene chip technology have been rapidly developed, and the development of these related technologies is also used as a premise and a foundation for implementing efficient breeding methods to improve reproductive performance of sows (Hickey et al, 2017), and the evaluation finds that filling SNP molecular marker chip data with whole genome sequencing data can improve accuracy of related analyses such as genome selection (Li Yong, et al, 2022). At present, whole genome association analysis has become one of important means for marker assisted breeding, and important mutation sites for controlling sow reproductive traits are screened out from a whole genome range through association analysis, so that theoretical basis is provided for revealing related genetic mechanisms, and the purposes of assisting breeding and improving sow reproductive performance are achieved (Zhao Desheng and the like, 2018). However, most studies on sow reproductive traits have been focused on increasing litter size, while at the same time easily ignoring the survival rate of piglets at birth. Ding et al performed genome-wide association analysis of the nest traits of Duroc meiosis and Duroc add, based on gene chip data, found that there was a significantly associated pleiotropic SNP on chromosome 7, affecting the number of stillbirth and primary piglet survival of Duroc meiosis (Ding et al 2021). The analysis of the genome-wide association of the number of stillbirth and the number of papillae in large white pigs by veraro et al resulted in 18 SNPs significantly correlated with the number of stillbirth and found 7 candidate genes affecting the number of stillbirth in large white pigs (veraro et al 2016).
The invention uses a uvlmm analysis module in GMAT software to carry out whole genome association analysis (Wang et al 2020) and screens SNP molecular marker loci which are obviously related to the characteristics of the stillbirth of the sow.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, performs genotyping by utilizing a gene chip technology, performs whole genome association analysis on the genetic chip technology and the sow stillbirth traits, screens out 6 SNP (single nucleotide polymorphisms) obviously related to the sow stillbirth traits, and provides new SNP molecular marker resources for the sow reproductive traits.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the applicant obtains SNP molecular markers obviously related to the characteristics of the stillbirth of the sow through whole genome association analysis screening, and obtains a nucleotide sequence of 100bp upstream and downstream of each SNP according to the 11.1 version of reference genome of the pig of the Ensmble database, which is specifically as follows:
the nucleotide sequence containing the SNP1 marker is shown as SEQ ID NO.1 and 2, the SNP1 marker is positioned at the 101 th position of the sequence and corresponds to the 68424849 th position of the chromosome 15 of the pig genome, the corresponding gene is SLC4A10, and the polymorphic site is C or T; when the genotype of the locus is TT, the sow has high stillbirth rate;
the nucleotide sequence containing the SNP2 marker is shown as SEQ ID NO.3 and 4, the SNP2 marker is positioned at the 101 th position of the sequence and corresponds to the 68442233 th position of the chromosome 15 of the pig genome, the corresponding gene is SLC4A10, the accession number in the dbSNP database is rs341306021, and the polymorphic site is T or C; when the genotype of the locus is CC, the sow has high stillbirth rate;
the nucleotide sequence containing the SNP3 marker is shown as SEQ ID NO.5 and 6, the SNP3 marker is positioned at the 101 th position of the sequence and corresponds to the 68516011 th position of the chromosome 15 of the pig genome, the corresponding gene is SLC4A10, and the polymorphic site is G or A; when the genotype of the locus is AA, the sow has high stillbirth rate;
the nucleotide sequence containing the SNP4 marker is shown as SEQ ID NO.7 and 8, the SNP4 marker is positioned at the 101 th position of the sequence and corresponds to the 68547427 th position of the chromosome 15 of the pig genome, the corresponding gene is SLC4A10, the accession number in the dbSNP database is rs318344759, and the polymorphic site is G or A; when the genotype of the locus is AA, the sow has high stillbirth rate;
the nucleotide sequence containing the SNP5 marker is shown as SEQ ID NO.9 and 10, the SNP5 marker is positioned at the 101 th position of the sequence and corresponds to the 68585156 th position of chromosome 15 of the pig genome, the corresponding gene is SLC4A10, and the polymorphic site is G or A; when the genotype of the locus is AA, the sow has high stillbirth rate;
the nucleotide sequence containing the SNP6 marker is shown as SEQ ID NO.11 and 12, the SNP6 marker is positioned at the 101 th position of the sequence and corresponds to the 69126372 th position of chromosome 15 of the pig genome, the corresponding gene is SLC4A10, and the polymorphic site is A or T; when the genotype of the locus is TT, the sow has high stillbirth rate.
Drawings
Fig. 1: the manhattan diagram made by example 2 visualizes the results of the GWAS analysis. There are 13 SNPs above the threshold line, one SNP on each of chromosome 5 and 7 is lncRNA, and 4 SNPs on chromosome 18 are located in intergenic regions, belonging to non-coding regions. The rest 7 SNP molecular markers which are located in the gene coding region and are remarkably related to the characteristics of the dead fetus of the large white sow and are screened by the invention.
Fig. 2: the QQ plot produced in example 2 was used to further determine the reliability of GWAS results by comparing the actual-log (P) values with the quantiles of the expected-log (P) value probability distributions to compare the two probability distributions.
Detailed Description
Example 1 genotyping detection and data processing
(1) DNA extraction
(1) Collecting ear tissues of 1175 white sows, mashing the collected ear-like tissues, placing the mashed ear-like tissues in a glass homogenizer, adding an equal volume of cell lysis buffer prepared from 100mmol/L Tris saturated phenol, 500mmol/L disodium ethylenediamine tetraacetate (EDTA), 20mmol/L sodium chloride (NaCL), 10% concentration Sodium Dodecyl Sulfate (SDS) and 20 mug/mL pancreatic RNase, adding 10ng/mL proteinase K, uniformly mixing, and placing the mixture in a 65 ℃ constant-temperature water bath for 30min;
slowly shaking the centrifuge tube for 15min, centrifuging at 12000rpm for 5min, and collecting supernatant
The liquid is put into another centrifuge tube;
(2) adding equal volumes of phenol, chloroform and isoamyl alcohol (the volume ratio is 25:24:1), shaking and mixing uniformly, placing in a centrifuge, centrifuging at 12000rpm for 5min, and taking supernatant into another centrifuge tube;
(3) adding equal volumes of phenol, chloroform and isoamyl alcohol (the volume ratio is 25:24:1), shaking and mixing uniformly, placing into a centrifuge, centrifuging at 12000rpm for 10min, and taking supernatant into another centrifuge tube;
(4) adding 2 times of pre-cooled absolute ethyl alcohol, standing until the ethyl alcohol volatilizes, picking out DNA precipitate and using ultrapure water to dissolve DNA;
(5) DNA quality was measured by DNA concentration meter and agarose gel electrophoresis.
(2) Genotyping assays
(1) Genotyping was performed using 50K SNP whole genome chips developed by Illumina company, which contain more than 50000 SNP molecular marker sites, according to the DNA extracted as described above;
(2) based on 152 white sow whole genome re-sequencing data, carrying out genotyping treatment on sequencing original data, and carrying out quality control on obtained SNP molecular marker loci by using PLINK v1.9 software, removing SNPs with detection rate smaller than 90% and minimum allele frequency smaller than 0.05, and finally using 17985419 SNPs for genotype filling.
(3) Genotype filling
And (3) filling chip data by using Beagle v5.1 software and taking large white pig whole genome re-sequencing data as a reference group, and screening 10197628 SNP molecular marker loci with filling accuracy higher than 0.8 for subsequent analysis.
EXAMPLE 2 Whole genome association analysis of SNP molecular markers and the stillbirth trait of white sows
The sequence and whole genome association analysis result in the invention is based on the 11.1 version information of the pig genome
(1) Phenotypic definition
The dead fetus of the sow is regarded as a case-control character, two or more dead fetuses of the sow are taken as cases according to the condition of dead piglets of each sow in the original phenotype record, the dead fetus of the sow is not taken as a control, and the case-control constructs a new phenotype for subsequent whole genome association analysis.
(2) SNP molecular marker quality control
And respectively carrying out quality control on the filled case phenotype and control phenotype SNPs by using PLINK v1.9 software according to the reliably SNP molecular marker loci screened after genotype filling, eliminating SNPs with minimum allele frequency less than 0.01 aiming at the case phenotype SNP data, eliminating SNPs with minimum allele frequency less than 0.01 aiming at the control phenotype SNP data, and merging and taking completely overlapped SNP molecular markers. Finally there were 1175 samples and 9409373 SNPs for whole genome association analysis.
(3) Whole genome association analysis of stillbirth traits of large white sow
The experimental herd used for whole genome association analysis in this example was a large white pig, including 1175 sows. According to the stillbirth information recorded by the experimental pig group, a method based on a mixed linear model is used for carrying out whole genome association analysis on SNP molecular markers and stillbirth traits of the large white sow by using a uvlmm analysis module in GMAT software. The specific model is as follows:
y＝μ+Wα+xβ+Zu+e
wherein y is a phenotype vector; mu is the population mean; w is a fixed effect design matrix; alpha is a classification variable of a fixed effect vector; x is the SNP vector; beta is SNP effect; z is a design matrix; u is the individual effect; e is the random residual.
According to the result of GWAS, the P value of each SNP locus is calculated as-log (P), and according to the calculation formula-log (1/SNP number) of a significant threshold line, the calculation of the invention shows that when the P value of each SNP locus is larger than or equal to 6.973561, the significant correlation exists between the SNP and the trait, and the manhattan diagram and the QQ diagram (figures 1 and 2) are painted by using the CMplot of R language.
Table 1: candidate SNP locus of white sow antenatal stillbirth character based on GWAS identification
Table 2: genotype frequency and stillbirth rate distribution of candidate SNP loci in white sows
Table 2 illustrates: the bracket indicates that the genotype sow has the frequency of 2 or more stillbirth.
As shown in table 1, 7 SNP sites significantly associated with the characteristics of the stillbirth of the sow were selected, the number of individuals of different genotypes at each SNP site and the number of individuals of stillbirth of 2 or more corresponding genotypes were further counted, and the stillbirth rate (the number of individuals of 2 or more stillbirth/the number of individuals of the genotype) of different genotypes was calculated. As can be seen from Table 2, for the 6 SNP loci of 15:68424849, 15:68442233, 15:6855011, 15:68557475, 15:6855156, 15:69126372, the frequency of producing dead fetus in sow is higher than that of other genotypes when the genotypes are TT, CC, AA, AA, AA, TT respectively; for the SNP site of 7:93672884, since individuals with recessive homozygous AA genotypes did not appear in 1175 sows, it was not possible to determine which genotypes affected the frequency of stillbirth in the sows.
Main references:
[1] li Yong, yang Manman, miao Zepu, shen Junran, chen Tao, wei Jiang. Application of low depth whole genome sequencing in large white pig reproductive trait genome selection [ J ]. Protect on agricultural biotechnology, 2022, 30 (02): 325-334.
[2] Wang Yuan analysis of genome-wide association of porcine reproductive trait and detection of copy number variation [ D ]. University of agriculture, china, 2018.
[3] Zhao Desheng, zhang Chongzhi. Research progress for genome-wide association analysis of livestock [ J ]. Chinese animal husbandry, 2018, 45 (02): 463-470.
[4]Ding R,Qiu Y,Zhuang Z,Ruan D,Wu J,Zhou S,Ye J,Cao L,Hong L,Xu Z,Zheng E,Li Z,Wu Z,Yang J.Genome-wide association studies reveals polygenic genetic architecture of litter traits in Duroc pigs[J].Theriogenology.2021,173:269-278.
[5]Hickey JM,Chiurugwi T,Mackay I,Powell W.Genomic prediction unifies animal and plant breeding programs to form platforms for biological discovery[J].Nat Genet,2017,49:1297-1303.
[6]Verardo LL,Silva FF,Lopes MS,Madsen O,Bastiaansen JW,Knol EF,Kelly M,Varona L,Lopes PS,SE.Revealing new candidate genes for reproductive traits in pigs:combining Bayesian GWAS and functional pathways[J].Genet Sel Evol,2016,48:9.
[7]Wang D,Tang H,Liu JF,Xu S,Zhang Q,Ning C.Rapid epistatic mixed-model association studies by controlling multiple polygenic effects[J].Bioinformatics,2020,36:4833-4837.
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Claims (1)
- The application of SNP markers in sow stillbirth trait selection is characterized in that the SNP markers are combined as follows:the nucleotide sequence containing the SNP1 marker is shown as SEQ ID NO.1 and 2, the SNP1 marker is positioned at the 101 st position of the sequence, and the polymorphic site is C or T;the nucleotide sequence containing the SNP2 marker is shown as SEQ ID NO.3 and 4, the SNP2 marker is positioned at the 101 st position of the sequence, and the polymorphic site is T or C;the nucleotide sequence containing the SNP3 marker is shown as SEQ ID NO.5 and 6, the SNP3 marker is positioned at the 101 st position of the sequence, and the polymorphic site is G or A;the nucleotide sequence containing the SNP4 marker is shown as SEQ ID NO.7 and 8, the SNP4 marker is positioned at the 101 st position of the sequence, and the polymorphic site is G or A;the nucleotide sequence containing the SNP5 marker is shown as SEQ ID NO.9 and 10, the SNP5 marker is positioned at the 101 st position of the sequence, and the polymorphic site is G or A;the nucleotide sequence containing the SNP6 marker is shown as SEQ ID NO.11 and 12, the SNP6 marker is positioned at the 101 st position of the sequence, and the polymorphic site is A or T;when the genotype of the SNP1 marker is TT, when the genotype of the SNP2 marker is CC, when the genotype of the SNP3 marker is AA, when the genotype of the SNP4 marker is AA, when the genotype of the SNP5 marker is AA, and when the genotype of the SNP6 marker is TT, the sow has high stillbirth rate, the pig breed is a white pig, and the stillbirth rate is that of the sow with two or more stillbirth rates.
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| CN113736889A (en) * | 2021-07-30 | 2021-12-03 | 华南农业大学 | SNP molecular marker related to pig stillbirth number and survival rate on pig chromosome 7 and application thereof |
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| CN108504752A (en) * | 2018-06-26 | 2018-09-07 | 华中农业大学 | A kind of and the associated molecular labeling of sow reproductive trait and application |
| CN113736889A (en) * | 2021-07-30 | 2021-12-03 | 华南农业大学 | SNP molecular marker related to pig stillbirth number and survival rate on pig chromosome 7 and application thereof |
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