CN112779339A - SNP molecular marker related to total papilla number of large white pigs as well as acquisition and application thereof - Google Patents
SNP molecular marker related to total papilla number of large white pigs as well as acquisition and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker related to the total papilla number of white pigs and acquisition and application thereof, wherein the SNP molecular marker acquires ear samples of the white pigs, extracts DNA, performs quality detection, performs genotyping to obtain SNP genotyping data, and obtains a molecular marker related to the total papilla number of the white pigs, and the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO: 1, an A/G allelic mutation exists at the 101 th base of the sequence, the nucleotide polymorphism of the sequence is caused, and when the 101 th nucleotide on the nucleotide sequence is G, the large white pig has more total papilla number.
Description
Technical Field
The invention relates to the fields of molecular biomarker technology and genetic breeding, in particular to an SNP molecular marker related to the total number of nipples of a white pig and acquisition and application thereof.
Background
The quantitative character of the number of pig teats is one of the important indexes for breeding the breeding performance of the breeding pigs, and has stronger correlation with the number born and the survival rate of piglets (Wiesner E, Willer S.Problems of inverted nipples in swine [ J ]. Monatsheftef fur Veterinarmedizin, 1978,33 (5): 189-. In addition, research shows that the variation coefficients of the number of the papillae of the American line and the Chinese line of the white pig are 4.24 percent and 4.18 percent respectively, the number of the papillae is relatively stable heredity (Zhao Zong, Yang Jing, Li Da quan, and the like. research on the heredity characteristics of the number of the papillae of the white pig and the black pig [ J ]. China livestock raising journal, 1999, 35 (6): 31-33.), and the breeding character breeding by taking the number of the papillae as an auxiliary character has reliability. Therefore, the method for improving the litter size and the survival rate of the pigs by breeding the pig breast number has great significance for improving the economic benefit of the pig industry.
Genome-wide association study (GWAS) is a method which integrates genetics, statistics and computer science, utilizes high-density molecular markers screened in a genome-wide range to scan a population to be researched in the genome-wide range, analyzes the association between the molecular marker data obtained by scanning and phenotypic traits by a statistical method, and then analyzes and identifies genetic variation of the phenotypic traits or related candidate genes influencing the phenotypic traits by combining genetic knowledge. The method is widely applied to breeding and production of livestock and poultry in recent years (Zhao, Pu bin, Guanwei, Zhaoqian, Mayuhui, pig important character complete genome association analysis research progress [ J ]. animal husbandry veterinary academy 2015, 46 (06): 873) 881.; Wang Wenhao, Zhang Haoxi, Wangjin jade, Limonihui, Wang Yongjuan, Jinghai yellow chicken gamma-interferon level complete genome association analysis [ J ]. Chinese animal husbandry veterinarian 2015, 42 (02): 245-.
The present patent uses 488 big white pig ear sample, extracts DNA and detects quality, carries out gene typing by a GeneSeek Port 50K SNP high density chip to obtain genotype data, uses MVP package of R language (X Liu et al. objective use of Fixed and Random Effect Models for Power and Efficient Genome-Wide Association students [ J ]. Plos Genetics, 2016, 12 (2): e1005767., YIN L, Zhang H, Tang Z, et al. rmvp: A mean-effective, visualization-enhanced, and parallel-authenticated. for Genome-Wide Association [ J ]. bioRxiv, 2020), combines MLM, GLM and the model, screens out pig model and the papillary, has important significance for pig total molecular marker, and provides important pig breeding for increasing the total pig molecular weight. The invention carries out GWAS analysis on the total nipple number character of the white pig by a high-density chip, and obtains an SNP molecular marker related to the total nipple number character of the white pig by screening, wherein the SNP molecular marker is obviously related to the total nipple number of the white pig, thereby providing a reference basis for the breeding of subsequent breeding pigs and the improvement of reproductive performance.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to the total papilla number of white pigs and acquisition and application thereof. The SNP molecular marker Affx-115258151 provided by the invention is 103221487 nucleotide sites of chromosome 7 of Sscrofa11.1 version 7 of the international white pig reference genome, and the polymorphism of the site base is A or G. In addition, the invention provides upstream and downstream primer sequences for detecting the site, so as to carry out high-efficiency detection. By optimizing the dominant allele of the SNP molecular marker and quickly detecting the locus, the invention can screen the SNP related to the total nipple number of the white pig, and provides a new molecular marker resource and marker-assisted selection application basis for the genetic breeding of the white pig.
In order to achieve the purpose, the invention adopts the technical scheme that:
an SNP molecular marker related to the total papilla number of the white pig, wherein the SNP molecular marker is Affx-115258151, and is 103221487 nucleotide sites of chromosome 7 of Sscrofa11.1 version 7 of the international white pig reference genome, bases of the sites are A or G, and the sites correspond to nucleotide sequences in a nucleic acid sequence table SEQ ID NO: 1, 101 th nucleic acid site.
The invention provides a primer for amplifying the SNP molecular marker or a probe for identifying the SNP molecular marker.
The invention provides a kit containing the primer or the probe.
The invention provides an application of the SNP molecular marker in white pig genetic breeding.
The invention provides a method for breeding a white pig variety or strain with high/low total papilla number characters by applying the SNP molecular marker, which comprises the following steps: extracting genome DNA of the large white pig, detecting the deoxynucleotide at 103221487 th site of chromosome 7, detecting the deoxynucleotide at 103221487 th site as A or G, determining that the genotype of the large white pig to be detected is AA type, AG or GG type, and selecting the large white pig with the AA type, AG or GG type genes according to breeding requirements for further seed selection and/or breeding, wherein the total number of the large white pig head with the GG type genes is far higher than the AA type and the AG type.
The invention provides application of the kit in breeding/assisted breeding of a white pig variety or strain with high total papilla number.
The invention provides a method for acquiring SNP molecular markers related to the total papilla number of white pigs, which comprises the following steps: extracting total DNA of ear tissues of the white pig, and performing quality control and analysis on the total DNA to obtain genotype data; counting the data of the total number of the individual nipples of the big white pig as a phenotype, and obtaining SNP (single nucleotide polymorphism) which is obviously associated with the total number of the nipples of the big white pig by utilizing a whole genome association analysis technology; the quality control standard is as follows: the detection rate of removed individuals is more than or equal to 90 percent, the detection rate of SNP is more than or equal to 90 percent, the frequency of minimum allele is more than or equal to 0.05, and the p value of Hardy-Weinberg equilibrium is more than or equal to 10-7(ii) a The whole genome association analysis adopts a mixed linear model as follows: y isijklmn＝μ+Gi+HYSj+Linek+Parityl+IDm+εijklmn
Wherein, the m is the original phenotype value of the total papillary number character of the m individual; u is the population mean; giIs Affx-115258151 molecular marker genotype effect (fixed effect); HYSjIs the combined effect (fixed effect) of the birth field, the birth year and the birth season; linekIs a strain effect (fixed effect); partylIs the birth fetal secondary effect (fixed effect); IDmIs an individual additive effect (random effect) that is assumed to follow a normal distribution:
representing individual effect variance; epsilonijklmnIs the model residual effect, obeying a normal distribution:
representing the residual variance.
The invention has the beneficial effects that:
the invention screens and determines the molecular marker of SNP related to the total nipple number of the white pig, provides the molecular marker and a kit for identifying SNP related to the total nipple number of the white pig, finally establishes a high-efficiency and accurate molecular marker assisted breeding technology, and applies the molecular marker assisted breeding technology to genetic improvement of the white sow for improving the total nipple number, thereby being capable of rapidly and accurately breeding the white pig, accelerating the breeding process, improving the reproductive performance of offspring white pigs, improving the enterprise profit and increasing the core competitiveness.
By detecting Affx-115258151 marker genotype to assist in breeding the large white sows with multiple papillae, the total number of the papillae can be increased and the nutrition level of the young large white sows can be improved by selecting and reserving core group large white sows homozygous for GG.
The method can detect the genotype of the white pig by adopting a gene chip technology in vitro, is used for evaluating the number of the papillae of the white pig for non-diagnosis purposes, and has the outstanding advantages of simplicity, rapidness, high sensitivity, good specificity and the like compared with the conventional PCR-RFLP method and the like.
Drawings
FIG. 1 is a Manhattan diagram of the present invention. Description of reference numerals: relating to the total papilla number character of the large white pig, the black arrow points to the marked SNP molecular marker Affx-115258151 screened by the invention, and the marker is positioned on the chromosome 7 of the large white pig.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
1. phenotypic-pedigree data acquisition
The basic study group for this experiment was a large white sow from a pig farm from a division of Guangxi GmbH. The complete pedigree comprises 14 generations of 7342 big white pigs, wherein phenotype data such as the number of left nipples, the number of right nipples, the number of umbilical prothesis, the number of small nipples, the number of blind nipples and the like of the 7342 big white pigs in 2019 and 2020 years are recorded. Total nipple number calculation formula: total number of papillae is obtained as the number of left papillae + the number of right papillae. After filtering the abnormal phenotype data, a total of 7342 observations of total papillary counts were obtained for phenotype-genotype correlation analysis.
2. Genotyping and quality control
(1) Ear tissue samples of 488 white sows were collected, DNA was extracted, and genotyping was performed using GGP 50k SNP (GeneSeek, US) chips to obtain 50697 SNP markers covering the whole genome.
(2) For all SNP markers located on 18 autosomes, quality control was performed using the Plink1.9 software, with the criteria: the individual detection rate is more than or equal to 90 percent; the SNP detection rate is more than or equal to 90 percent, the minimum allele frequency is more than or equal to 0.05, and the Hardy-Weinberg equilibrium p value is more than or equal to 10-7. For deletion genotypes, filling was performed using beagle5.1 software.
Based on the above quality control criteria, the remaining 482 and 38823 SNP markers were used for genome-wide association analysis, with 482 large white pigs having both total papillary number phenotype data and genotype data.
3. Whole genome association analysis of total papilla number of large white pigs
And (3) carrying out GWAS analysis by using an MLM model, a GLM model and a farmCPU in an MVP software package under the R statistical environment, respectively obtaining SNP markers which are detected by various methods and are obviously associated with the total nipple number traits, and taking the SNP markers which are detected by at least two methods as candidate SNPs.
For p values of all markers, -log10 is taken to draw a Manhattan graph (figure 1), and three analysis methods such as MLM, GLM and farmCPU models are combined to display and screen the SNP markers with significant association.
As shown in fig. 1, in large white pigs, there are sites in chromosome 7 that significantly affect the effective total papilla number.
4. Correlation analysis of Affx-115258151 molecular marker typing result and total nipple number
The correlation analysis of Affx-115258151 molecular marker and the total number of papillae of white American big pigs was performed using Mixed Linear Model (MLM). The concrete model is as follows:
Yijklmn＝μ+Gi+HYSj+Linek+Parityl+IDm+εijklmn
wherein, is the m-thOriginal phenotypic value of total papillary number character; u is the population mean; giIs Affx-115258151 molecular marker genotype effect (fixed effect); HYSjIs the combined effect (fixed effect) of the birth field, the birth year and the birth season; linekIs a strain effect (fixed effect); partylIs the birth fetal secondary effect (fixed effect); IDmIs an individual additive effect (random effect) that is assumed to follow a normal distribution:
representing individual effect variance; epsilonijklmnIs the model residual effect, obeying a normal distribution:
representing the residual variance. The differences in total papillary number traits among the three genotype individuals were significantly analyzed using the F-test, and the results of the analysis are shown in table 1.
TABLE 1 influence of Affx-115258151 marker on Total Nipple number in white pig
Table 1 illustrates: the difference is obvious when P is less than 0.05; p < 0.01 is very different.
As can be seen from Table 1, the molecular marker has a higher average total papilla number in the Affx-115258151 marker locus on chromosome 7 than in the AA type and the AG type, indicating that the homozygote GG type is advantageous for the average total papilla number. The GG type is extremely obvious compared with the AA type and the GG type is extremely obvious compared with the AG type, which indicates that the allele G is favorable for the total number of papillae. The total number of nipples is an important index for measuring the reproductive performance of the big white pigs, and the higher total number of nipples indicates that the reproductive performance of the big white pigs is better. Therefore, the elimination of AA genotype big white pigs can bring more economic benefits, and the elimination of AA genotype big white sows and the retention of GG type and big white sows are needed in the breeding process to improve the frequency of the allele G of the locus generation by generation.
Sequence listing SEQ ID NO: 1 is the 100bp nucleotide sequence of the upstream and downstream of SNP molecular marker Affx-115258151 which is screened and associated with the total number of nipples of the white pig, the length of the nucleotide sequence is 201bp, and an A/G allelic gene mutation exists at the R position of the 101 th base of the sequence. This mutation can cause the mutation of SEQ ID NO: 1 to produce a polymorphism.
Example 2:
according to the gene result obtained by screening in the above example 1, the SNP molecular marker Affx-115258151 related to the total number of papillae of the white pig is shown, the molecular genetic marker is located at the 103221487 nucleic acid site of the 7 th chromosome of the white pig, and the position is a G > A mutation and corresponds to the 101 st nucleic acid site of the nucleic acid sequence table SEQ ID N0.1.
Example 3:
the skilled person can easily design primers for amplifying the molecular markers or probes for identifying the molecular markers with reference to the molecular genetic markers according to the present invention, and then use the primers or probes for detecting the molecular markers, such as obtaining the molecular genetic markers by PCR amplification and obtaining corresponding sequences by clone sequencing, or detecting the Bsm-RFLP polymorphism. Thus, the invention also includes primers for amplifying the molecular genetic marker or probes for identifying the molecular genetic marker, and kits containing the primers or probes.
Example 4:
the molecular genetic marker can be applied to assist in breeding or assisting in breeding of the white pigs, and the specific method comprises the following steps: extracting genome DNA of the big white pig, detecting the 103221487 th deoxynucleotide of the 7 th chromosome, and detecting that the 103221487 th deoxynucleotide is A or G; judging whether the genotype of the big white pig to be detected is AA type, AG type or GG type according to the locus genotype; according to breeding requirements, the large white pig with AA type, AG type or GG type genes is selected for further breeding and/or breeding. The total number of nipples of the homozygous GG type large white pigs is higher than that of the AA type large white pigs, so that more economic benefits can be brought by eliminating the AA genotype large white pigs, the AA genotype large white pigs need to be eliminated in the breeding process, and the GG type large white pigs are reserved to improve the frequency of the allele G of the site generation by generation.
As can be seen from Table 1, the total head number of GG type large white pigs is 0.7 higher than that of AA type, 7000 piglets can be suckled by 10000 large white sows capable of breeding per fetus, the piglets are directly fattened to 100kg for marketing calculation, 595 tons of long white pork can be provided by ten thousand large white sows according to the estimation of the dressing percentage of 85 percent, which brings huge economic income and effect to enterprises and increases the core competitiveness of the enterprises.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Sequence listing
<110> Guangxi Yangxi agriculture and animal husbandry Limited liability company
Guangxi Yangxi Xiang pig Gene science and technology Co Ltd
Guangxi Guibour Xibo Gene science and technology Co Ltd
Guigang Ruikang fodder Co Ltd
<120> SNP molecular marker related to total papilla number of large white pigs and acquisition and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> DNA
<213> genus swine (Sus scrofa)
<220>
<221> misc_feature
<222> (101 )..( 101)
<223> r is a or g
<400> 1
tgaaaagaag caggaaatac caagaaacct tttttttttc ctccaaaaga tttcctggcc 60
acattgccag atttgactct aaaagtaaat tgtccacttc rgtgaaatat ttaaacgttt 120
tgtcttccct tccttcaaag gcaaactaag ctggtgaaga agcaagggta gggaagcttt 180
gcttggggta gggcacgaag g 201
Claims (7)
1. An SNP molecular marker related to the total papilla number of the white pig, which is characterized in that the SNP molecular marker is Affx-115258151, and is 103221487 nucleotide sites of chromosome 7 of international white pig reference genome Sscrofa11.1 version 7, the base of the site is A or G, and the site corresponds to a nucleotide sequence shown in a nucleic acid sequence table SEQ ID NO: 1, 101 th nucleic acid site.
2. A primer for amplifying the SNP molecular marker of claim 1 or a probe for identifying the SNP molecular marker of claim 1.
3. A kit comprising the primer or probe of claim 2.
4. Use of the SNP molecular marker according to claim 1 in the genetic breeding of white pigs.
5. A method for breeding/assisting in breeding a white pig variety or line with high/low total papillary number characters by using the SNP molecular marker of claim 1, wherein the method comprises the following steps: extracting genome DNA of the large white pig, detecting the deoxynucleotide at 103221487 th site of chromosome 7, detecting the deoxynucleotide at 103221487 th site as A or G, determining that the genotype of the large white pig to be detected is AA type, AG or GG type, and selecting the large white pig with the AA type, AG or GG type genes according to breeding requirements for further seed selection and/or breeding, wherein the total number of the large white pig head with the GG type genes is far higher than the AA type and the AG type.
6. Use of the kit according to claim 3 for breeding/assisted breeding of a white pig breed or line with high total papillary number.
7. Obtaining SNP (Single nucleotide polymorphism) score related to total papilla number of large white pigsA method of sub-tagging, the method comprising: extracting total DNA of ear tissues of the white pig, and performing quality control and analysis on the total DNA to obtain genotype data; counting the data of the total number of the individual nipples of the big white pig as a phenotype, and obtaining SNP (single nucleotide polymorphism) which is obviously associated with the total number of the nipples of the big white pig by utilizing a whole genome association analysis technology; the quality control standard is as follows: the detection rate of removed individuals is more than or equal to 90 percent, the detection rate of SNP is more than or equal to 90 percent, the frequency of minimum allele is more than or equal to 0.05, and the p value of Hardy-Weinberg equilibrium is more than or equal to 10-7(ii) a The whole genome association analysis adopts a mixed linear model as follows: y isijklmn＝μ+Gi+HYSj+Linek+Parityl+IDm+εijklmn
Wherein, the m is the original phenotype value of the total papillary number character of the m individual; u is the population mean; giIs the Affx-115258151 molecular marker genotype effect; HYSjIs the combined effect of the birth field, the birth year and the birth season; linekIs a strain effect; partylIs the effect of birth times; IDmIs an additive effect in individuals, assumed to follow a normal distribution:
representing individual effect variance; epsilonijklmnIs the model residual effect, obeying a normal distribution:
representing the residual variance.
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| CN114574593A (en) * | 2022-01-25 | 2022-06-03 | 湖北省农业科学院畜牧兽医研究所 | Application of pig SNP molecular marker in pig breast number character screening and pig breeding |
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| CN114574593A (en) * | 2022-01-25 | 2022-06-03 | 湖北省农业科学院畜牧兽医研究所 | Application of pig SNP molecular marker in pig breast number character screening and pig breeding |
| CN114574593B (en) * | 2022-01-25 | 2023-09-22 | 湖北省农业科学院畜牧兽医研究所 | Application of pig SNP molecular marker in pig nipple number character screening and pig breeding |
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