CN108893544B

CN108893544B - SNP molecular marker related to litter size of pig menstruation, identification and application thereof

Info

Publication number: CN108893544B
Application number: CN201810613697.8A
Authority: CN
Inventors: 杨亚岚; 李奎; 周荣; 刘颖; 李文通; 李华; 于辉
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2018-06-14
Filing date: 2018-06-14
Publication date: 2021-11-16
Anticipated expiration: 2038-06-14
Also published as: CN108893544A

Abstract

The invention discloses an SNP locus related to the litter size of a pig menstruation, wherein the SNP locus is positioned at Chr7:75245594 of a pig reference genome Sscrofa11.1, and two allelic genes of the SNP locus are C and T; the three genotypes corresponding to the SNP loci are respectively CC, TT and TC, and compared with the pigs with the genotypes of TT and TC, the pigs with the genotypes of CC have higher parity number. The determination of the relevance of the SNP locus and the litter size of the high-yield pigs can be effectively used for identifying or assisting in identifying the high-yield pigs with the litter size and accelerating the breeding process of the high-yield pigs with the litter size.

Description

SNP molecular marker related to litter size of pig menstruation, identification and application thereof

Technical Field

The invention relates to the field of pig breeding, in particular to an SNP molecular marker related to the litter size of a pig warp and used for improving the pig breeding efficiency and identification thereof.

Background

Reproductive performance is one of the key factors affecting the economic benefit of the pig industry. As perennial animals, the litter size, especially the per-birth litter size, is an important index of reproductive traits of pigs, and the litter size can be directly related to the number of fattening pigs and the pork supply.

The reproductive capacity of sows is affected by a number of factors, including sow breed, parity, placental efficiency, hormone levels, nutritional levels, mating work, etc. Among them, steroid hormones including adrenocortical hormone and sex hormones (male hormone and female hormone) play important roles in regulating the reproductive capacity of sows and maintaining normal pregnancy. The cognition of the action and the molecular mechanism of the hormone synthesis, particularly the exploration of the action of a new regulatory gene or enzyme in the hormone synthesis process and the influence on the reproduction, has very important guiding significance for guiding the application of exogenous reproductive hormone in the reproduction production process, improving the reproduction production efficiency and breeding the sows with high fertility. Meanwhile, the identification of genes related to the litter size of the born pig can provide important clues for explaining the genetic mechanism of the growth and development of the fetus of the pig and other mammals and provide theoretical basis for the genetic improvement of the reproductive traits of the pig.

Finding out the relation between genes and traits through correlation analysis between the genes and traits is an important means for researching gene functions. At present, the most widely used method for screening related SNP sites (single nucleotide polymorphism sites) is genome-wide association analysis (GWAS). GWAS is a method for scanning a population to be researched by screening high-density molecular markers in a genome-wide range and analyzing the correlation between molecular marker data obtained by scanning and phenotypic traits. However, the method of GWAS is used for searching SNP sites related to the pig litter size trait, the obtained results are numerous and jumbled and disordered, and most of gene variation is not related to the target trait. It is quite difficult to select the relevant SNP site in the result of this bulky disorder. Therefore, it is necessary to select a specific gene in advance and determine the relevant SNP site located on the specific gene in combination with the GWAS method.

The current method for obtaining high-yield litter size pigs mainly focuses on genome editing, and realizes accurate modification of genomes by 'editing' target genes, including knockout of specific DNA fragments, introduction of specific mutations, transfer of site-specific DNA fragments and the like. However, people increasingly pay attention to the effect of pig breeding methods on pig quality and the influence on human health while the requirement on pork yield is improved. Because of the difficulty of the scientific community in verifying that transgenic animals have no side effects on human health in a short time and the insufficient cognition of the transgenic animals by the broad masses, people have a strong desire to eat non-transgenic animals. Apart from genome editing techniques, the realization of other breeding theories also requires knowledge of the pig genome. However, the understanding and recognition of the pig genome is far from sufficient, and the control of genes affecting the breeding efficiency of pigs is still insufficient, so that further research is necessary to meet the requirements of pig breeding methods.

Therefore, the invention aims to clarify a gene related to the litter size of the pig birth, provide an SNP molecular marker related to the litter size of the pig birth, the identification and the application of the SNP molecular marker, and search the SNP molecular marker related to the litter size traits of the pig by performing the association analysis of the gene polymorphism and the litter size of the pig birth on a specific gene; meanwhile, a brand-new breeding method of the pigs with high breeding efficiency is provided, the SNP molecular marker is used for breeding the pig species, and the promotion of the national pig breeding theory and technical level is realized.

Disclosure of Invention

Aiming at the problems in the pig breeding method, the inventor carries out intensive research, carries out association analysis on the polymorphism of the DHRS4 gene and important reproduction traits by using a whole genome association analysis method, finds out SNP molecular markers related to the litter size of the born pig gene for the first time, and enriches SNP sites influencing the pig reproduction efficiency; meanwhile, a multigroup chemical integration accurate breeding method for improving the breeding efficiency (the litter size of the produced pigs) of the pigs is also provided, the method does not adopt a genome editing means, improves the acceptance of the broad masses and can synthesize the multilayer genetic variation effect, thereby completing the invention.

The object of the present invention is to provide the following:

(1) an SNP site related to the litter size of swine, wherein the SNP site is positioned at Chr7:75245594 of a swine reference genome Sscrofa11.1, the NCBI reference SNP number is rs701332503, and two allelic genes of the SNP site are C and T.

(2) According to the SNP locus described in (1), the three genotypes corresponding to the SNP locus are respectively CC, TT and TC genotypes, and compared with the swine with the genotypes of TT and TC, the swine with the genotype of CC has higher litter size through birth.

(3) The SNP site according to (1) above, wherein the method for detecting genotyping of the SNP site involves a PCR amplification reaction in which an amplification primer P is present₁And P₂The nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

(4) The SNP site according to (1) above, wherein the method for detecting genotyping of said SNP site involves a single base extension reaction in which the nucleotide sequence of the extension primer is shown in SEQ ID NO. 3.

(5) The SNP site according to (1), wherein the kit for detecting the SNP site comprises a set of amplification primers P₁And P₂The amplification primer P₁And P₂The nucleotide sequences of (A) respectively comprise sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; and/or

The kit for detecting the SNP locus comprises an extension primer, wherein the nucleotide sequence of the extension primer comprises a sequence shown in SEQ ID NO. 3; and/or

The kit for detecting the SNP locus also comprises a PCR reaction reagent and a PCR product purification reagent.

(6) A method for identifying or assisting in identifying the litter size of a pig, the method comprises the steps of detecting whether deoxyribonucleotides at Chr7:75245594 of a pig reference genome Ssicrofa 11.1 are C, T or C and T to determine that the genotype of a pig to be detected is CC, TT or TC, and determining the litter size of the pig according to the genotype: compared with the pig with the genotype of TT or TC, the pig with the genotype of CC has higher birth cost,

wherein, the CC genotype is the homozygote of the DNA of the SNP locus of the pig which is C; the TT genotype is the homozygote of the pig with the deoxyribonucleotide of the SNP site as T; the TC gene type is a heterozygote of T and C which is the deoxyribonucleotide of the SNP site of the pig.

(7) A breeding method of high-yield litter size pigs, the method comprising the steps of selecting pigs of which genotype is CC at Chr7:75245594 of reference genome sscrofa11.1 as parents for breeding;

specifically, the breeding method comprises the following steps:

step 1, selecting a pig with a genotype CC at Chr7:75245594 of a reference genome Ssicrofa 11.1 as a parent for breeding to obtain an F1 generation piglet;

step 2, carrying out genotype analysis on the F1 generation piglets, breeding piglets with the genotype of CC at Chr7:75245594 of the reference genome Ssicrofa 11.1, and continuously breeding after the piglets grow up to obtain F2 generation piglets;

step 3, carrying out genotype analysis on the F2 generation piglets, breeding piglets with the genotype of CC at Chr7:75245594 of the reference genome Ssicrofa 11.1, and continuously breeding after the piglets grow up to obtain F3 generation piglets;

and (3) repeating the step (2) or the step (3) to finally obtain the breeding pig with genotype CC and high litter size heritability.

(8) The breeding method according to the above (7), further comprising determining a litter size of the produced pig by measuring an expression amount of mRNA of DHRS4 gene in the pig ovarian tissue;

wherein, the lower the expression quantity of the mRNA of the DHRS4 gene in the ovary tissue of the pig is, the higher the born number of the pig is, and the higher the expression quantity of the mRNA is, the lower the born number of the pig is.

(9) The breeding method according to the above (7), further comprising determining the number born by the pig by measuring the expression level of NRDR protein encoded by DHRS4 gene in the pig ovarian tissue;

wherein, the lower the expression level of the NRDR protein, the higher the born number of the pig, and the higher the expression level of the NRDR protein, the lower the born number of the pig.

(10) Use of the SNP site of (1) above for identifying or assisting in identifying litter size of swine;

the application of the expression quantity of the mRNA of the DHRS4 gene in the pig ovarian tissue in the aspect of identifying or assisting in identifying the litter size of the pig;

the application of the expression quantity of the NRDR protein coded by the DHRS4 gene in the pig ovarian tissue in the aspect of identifying or assisting in identifying the litter size of the pigs.

(11) The use of the amplification primer for detecting the SNP site of (3) above, or the extension primer for detecting the SNP site of (4) above in identifying or assisting in identifying the litter size of swine.

According to the SNP molecular marker related to the litter size of the swine parity, the identification and the application thereof, the invention has the following beneficial effects:

the inventor selects a specific DHRS4 gene from a plurality of genes to carry out pig breeding trait association analysis, discovers that the correlation exists between a specific SNP site in the gene and the born number for the first time, can be effectively used for identifying or assisting in identifying the high-yield born pigs and quickening the breeding process of the high-yield born pigs.

The SNP locus genotype analysis in the DHRS4 gene, the DHRS4 gene expression analysis and other multi-group chemical detection are combined, high-stability and high-yield pig seeds are obtained through artificial breeding on the premise of not adopting a genome editing means, the application of a multi-group chemical integration precise breeding theory is realized, and the national pig breeding theoretical level and technical level are obviously improved.

Drawings

FIG. 1 shows the difference in expression of DHRS4 gene mRNA levels in the ovaries of white pigs and Meishan pigs; (ii) a

FIG. 2 shows the difference in the expression of DHRS4 gene protein levels in ovaries in bletilla striata and Meishan pigs;

FIG. 3 shows the difference in the expression of DHRS4 gene protein levels in the ovaries of bletilla striata and Meishan pigs.

Detailed Description

The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In the invention, the large white pig is selected to carry out the identification of SNP loci related to farrowing characters. The white pig accounts for a higher proportion in the breeding of the pig breeds in China, and the selection of the pig breeds is convenient for the subsequent breeding of the pig breeds in China.

The invention uses a genome-wide association analysis (GWAS) method to search SNP sites related to the pig litter size traits. The GWAS method for searching the SNP locus related to the litter size of the pig has the disadvantages that a complicated and disordered result is easy to obtain, and most of gene variation is not related to target characters; there is great difficulty in selecting the relevant SNP site in this result of bulky disorder. Therefore, it is necessary to select a specific gene in advance and determine the relevant SNP site located on the specific gene by combining with the GWAS method.

Therefore, the specific gene is selected from a plurality of genes to be screened, so that the SNP locus on the gene is determined, and the success rate of determining the related SNP locus is greatly improved. Wherein the specific gene is DHRS4 gene.

Research shows that NRDR (coenzyme II dependent retinol resistant dehydrogenation/reductase) encoded by DHRS4 gene not only participates in vivo retinoic acid synthesis, but also shows different catalytic activities on steroid hormones, vitamin K3, isatin and other substances. The result of high-throughput detection of gene differential expression of embryonic development and tumor cells and normal cells shows that the DHRS4 gene is up-regulated in the development process of embryonic limbs. In this regard, the present inventors considered that the DHRS4 gene may be involved in intracellular signaling by affecting the synthesis of factors such as retinoic acid, and is involved in the occurrence of proliferative differentiation of embryonic cells.

Animal breeding trait research shows that the expression of DHRS4 gene in pig testis with high androgen is higher than that of pig testis with low androgen, but no other intensive research results are found after the discovery. The inventors consider that the DHRS4 gene is involved in the synthesis of steroid hormones. However, the mechanism of action of the specific DHRS4 gene in regulating steroid hormone synthesis is not clear and further studies are needed.

Among the existing studies, DHRS4 gene research has mainly focused on analyzing its expression in different tissues. The gene has less research in pigs, and the relationship between the gene and upstream and downstream genes in a breeding related pathway still needs to be disclosed in the aspects of a molecular mechanism for regulating and controlling the breeding performance of the pigs and the like. The inventor carries out association analysis on the polymorphism of the gene and important reproductive traits by using a whole genome association analysis method, and determines SNP sites related to litter traits in the gene for the first time through a large amount of researches.

The GWAS method is combined with the screened specific gene by the inventor to provide an SNP site related to the litter size of the pig in the DHRS4 gene, wherein the SNP site is positioned at Chr7:75245594 of a pig reference genome Scrofa11.1, the NCBI reference SNP number is rs701332503, and two alleles of the SNP site are C and T. The gene frequency of deoxyribonucleotide C was 0.432, and the gene frequency of deoxyribonucleotide T was 0.568.

Three genotypes corresponding to the SNP locus are respectively CC, TT and TC, and the CC genotype is a homozygote of the pig with the deoxyribonucleotide of the SNP locus as C; the TT genotype is the homozygote of the pig with the deoxyribonucleotide of the SNP site as T; the TC gene type is a heterozygote of C and T of deoxyribonucleotide at the SNP site of the pig.

The inventor finds that the pig with the CC genotype has higher born number compared with the pig with the TT and TC genotypes through a great deal of research.

In the invention, the genotyping utilizes matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology: firstly, PCR amplifies the target sequence, then adds SNP sequence specific extension primer, and extends 1 base on the SNP site. The prepared sample analytes were co-crystallized with the chip matrix and subjected to transient nanosecond (10) in a vacuum tube of a mass spectrometer^-9s) strong laser excitation, nucleic acid molecule desorption and conversion into metastable state ion, ion flight time in electric field is inversely proportional to ion mass, and detection of nucleic acid molecule in true state by flight time detectorThe precise molecular weight of the sample analyte is obtained by the flight time in the empty tube, so that the SNP locus information can be detected, and the method is also called as a Sequenom MassArray SNP genotype analysis technology.

As described above, the SNP site genotyping measurement involves steps such as extraction of DNA from pig genome, PCR amplification reaction, and single base extension reaction.

Wherein, the amplification primer P in the PCR amplification reaction₁And P₂The nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

Wherein the nucleotide sequence of the extension primer in the single base extension reaction is shown as SEQ ID NO. 3.

Correspondingly, the invention provides a detection method for SNP locus genotyping related to the litter size of the pig parity, which comprises the following steps:

(1) extracting the genomic DNA of the pig;

(2) and (3) taking the genome DNA of the pig to be detected as a template, and carrying out Sequenom MassArray detection by using an amplification primer and an extension primer to determine the genotype at the Chr7:75245594 of the reference genome Sscrofa11.1 of the pig.

Wherein the amplification primer (P) in the PCR amplification reaction₁And P₂) As shown in SEQ ID NO.1 and SEQ ID NO. 2; the extension primer is shown as SEQ ID NO. 3.

The detection method for the SNP locus genotyping related to the litter size of the swine menstruation products further comprises a direct sequencing method or a kit determination method.

The kit comprises (1) a PCR amplification primer P₁And P₂(ii) a Preferably, the kit also comprises (2) PCR reaction reagents: comprises PCR buffer solution, dNTP, PfuDNA polymerase and SNaPshot mixed solution; (3) PCR extension primer of SNP locus; (4) PCR product purification reagents: including exonuclease, shrimp-alkaline phosphatase, and buffer for purification.

The use method of the detection kit provided by the invention comprises the following steps: extracting a DNA sample from pig ear or other tissue cells; preparing a PCR reaction system, carrying out PCR amplification and purifying a PCR product; simultaneously carrying out extension reaction on the PCR product and the extension primer of the SNP point; performing capillary electrophoresis analysis on the extension product; SNP locus analysis, and further gene typing information can be obtained. The SNP site analysis can adopt, but is not limited to, an ABI3730XL automatic sequencer.

The invention provides a method for identifying or assisting in identifying the litter size of a pig, which comprises the steps of detecting that deoxyribonucleotide at Chr7:75245594 of a pig reference genome Ssicrofa 11.1 is C, T or C and T, determining that the genotype of the genetic locus is CC, TT or TC, predicting the litter size of the pig according to the genotype: pigs with genotype CC have a higher litter size than pigs with genotype TT or TC.

The invention provides a breeding method of high-yield litter size pigs, which comprises the step of selecting pigs with the genotype CC at Chr7:75245594 of a reference genome Sscrofa11.1 as parents for breeding. Specifically, the method comprises the following steps:

step 1, selecting a pig with a genotype CC at Chr7:75245594 of a reference genome Ssicrofa 11.1 as a parent (F0) for mating to obtain a first-generation (F1 generation) piglet;

step 2, carrying out genotype analysis on the F1 generation piglets, breeding piglets with the genotype CC at Chr7:75245594 of the reference genome Ssicrofa 11.1, and continuously breeding after the piglets grow up to obtain F2 generation piglets;

and 3, carrying out genotype analysis on the F2 generation piglets, breeding piglets with the genotype CC at the Chr7:75245594 of the reference genome Ssicrofa 11.1, and continuously breeding after the piglets grow up to obtain the F3 generation piglets.

And (3) repeating the step (2) or the step (3) to finally obtain the breeding pig with genotype CC and high litter size heritability. The method follows natural propagation and obtains the breeding pigs with high birth litter size through less manual intervention; meanwhile, the breeding method is a non-transgenic method, the acceptance of the masses is higher, the development of the pig industry can be effectively promoted, and the core competitiveness of the pig breeding industry in China is improved.

The invention also provides application of the SNP locus in identification or auxiliary identification of the litter size of the pig.

The invention also provides application of the extension primer or the amplification primer for detecting the SNP locus in the aspect of identification or auxiliary identification of the litter size of the born pig.

The invention also provides application of the genotyping method for detecting the SNP locus in the aspect of identifying or assisting in identifying the litter size of the born pig.

The inventor considers that: classical genetic breeding theory is based on "hard inheritance" (mendelian inheritance), which essentially only considers the influence of DNA sequence variants on shape genetic variation. The results of large-scale GWAS analysis show that the genome-wide related SNPs can only explain part of phenotypic variation, and part of the phenotypic variation cannot be determined by DNA-level variants, namely, the phenomenon of missing heritability exists. Although there have been attempts by scholars to correct the "missing heritability" problem from a statistical point of view, there is a lack of biologically sound interpretation. In the conventional concept, the process of transmission of genetic information from DNA to phenotype is almost linear, except for RNA editing, splicing, and protein molecular modification, and as for all links below DNA, including intermediate molecular information and phenotype, are ultimately controlled by genomic DNA, the heritable components of an organism are completely determined by the sequence information of genomic DNA. Therefore, pig genome selection and genome editing breeding are considered as important ways to break through the bottleneck of pig breeding.

However, with the progress of research, the conventional idea is being broken. It is recognized that the effect of DNA level variation on the phenotypic difference of pig groups and individuals is overestimated, the genome expression regulation mechanism is not completely clear, the effect of multilayer omics on the phenotype is underestimated, and the personalized genetic mechanism of the pig species is not concerned. The huge phenotypic genetic difference among the individual pigs depends not only on the variation of the genome structure and the DNA sequence, but also on the multi-level expression regulation of the genome. In order to realize the efficient application of genome information, it is necessary to develop a research on the multigroup variation of pigs, integrate multigroup information, establish a population personalized multigroup chemical integration precise breeding theory and technical system, and realize multigroup selection, which will significantly improve the breeding theory and technical level of pigs in China.

Based on the above considerations, the present inventors have analyzed and verified factors affecting pig production efficiency at the transcription level and the protein expression level.

The invention provides a method for identifying or assisting in identifying the litter size of a pig, which is used for determining the litter size of the pig by measuring the expression quantity of DHRS4 gene mRNA in the ovary tissue of the pig.

The method for identifying or assisting in identifying the piglet traits comprises the steps of obtaining total RNA of ovarian tissue cells of the pigs, determining the expression quantity of mRNA of DHRS4 gene of the pigs through reverse transcription and quantitative PCR, wherein the lower the expression quantity of the mRNA, the higher the born number of the pigs, and the higher the expression quantity of the mRNA, the lower the born number of the pigs.

Wherein, the quantitative PCR amplification procedure comprises the following steps: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 5 s; extending at 60 ℃ for 30 s; repeat 40 cycles; 95 ℃ for 15 s; 60 ℃ for 1 min; 95 ℃ for 15 s; 60 ℃ for 15 s.

Wherein, the PCR amplification primer P for measuring the expression quantity of DHRS4 gene mRNA₃And P₄，P₃And P₄The nucleotide sequences of (A) are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.

The invention also provides a kit for determining the expression quantity of DHRS4 gene mRNA, and the kit comprises an amplification primer P₃And P₄，P₃And P₄The nucleotide sequences of (A) are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.

Furthermore, the invention also provides a breeding method of pigs with high farrowing number, which comprises the step of selecting sows with low expression level of DHRS4 gene mRNA in the ovary tissues of the pigs as parents to breed. Specifically, the method comprises the following steps:

step 1, selecting a sow with low expression level of DHRS4 gene mRNA in an ovarian tissue as a parent (F0) for mating to obtain a first generation (F1 generation) piglet;

step 2, carrying out mRNA quantitative analysis on the F1 generation piglets, breeding piglets with low expression quantity of DHRS4 gene mRNA in ovarian tissues, and continuously breeding the piglets after the piglets grow up to obtain F2 generation piglets;

and 3, carrying out mRNA quantitative analysis on the F2 generation piglets, breeding piglets with low expression level of DHRS4 gene mRNA in ovarian tissues, and continuously breeding the piglets after the piglets grow up to obtain the F3 generation piglets.

Repeating the step 2 or 3 to finally obtain the breeding pig with low expression DHRS4 and high inheritance in the ovarian tissue.

The invention also provides application of the DHRS4 gene, the amplification primer, the expression quantity test method and the expression quantity detection kit which are related to the farrowing characters of the pigs in the aspect of identifying and/or breeding the breeding pigs on the basis of the disclosure, wherein the breeding pigs have higher farrowing number.

The invention provides a method for identifying or assisting in identifying the litter size born by pigs, which determines the litter size born by pigs by measuring the expression level of NRDR protein encoded by DHRS4 gene in ovary tissues of pigs.

The method for identifying or assisting in identifying the piglet traits comprises the steps of obtaining total protein of a porcine ovarian tissue cell, and determining the expression quantity of NRDR protein coded by DHRS4 gene in a porcine ovarian tissue through SDS-polyacrylamide gel electrophoresis, membrane transfer, antibody incubation and color development, wherein the lower the expression quantity of the NRDR protein is, the higher the born number of the pig is, and the higher the expression quantity of the NRDR protein is, the lower the born number of the pig is.

Further, the present invention provides a method for breeding a high-farrowing pig, comprising the step of selecting a sow having a low expression level of the NRDR protein encoded by the DHRS4 gene in ovarian tissue as a parent to breed. Specifically, the method comprises the following steps:

step 1, selecting a sow with low expression level of NRDR protein coded by DHRS4 gene in ovarian tissue as a parent (F0) for mating to obtain a first generation (F1 generation) piglet;

step 2, carrying out quantitative analysis on NRDR protein encoded by DHRS4 gene on F1 generation piglets, breeding piglets with low expression level of NRDR protein in ovarian tissues, and continuously breeding after the piglets grow up to obtain F2 generation piglets;

and 3, carrying out quantitative analysis on NRDR protein encoded by the DHRS4 gene on the F2 generation piglets, breeding piglets with low expression level of the NRDR protein in ovarian tissues, and continuously breeding the piglets after the piglets grow up to obtain the F3 generation piglets.

Repeating the step 2 or 3 to finally obtain the breeding pigs with low expression NRDR protein and high inheritance in the ovarian tissues.

In the invention, specifically, the multigroup chemical integration accurate breeding method comprises the following steps:

step 1, selecting sows meeting at least the following two conditions as parents (F0) for mating to obtain first-generation (F1 generation) piglets: (i) low expression level of mRNA of DHRS4 gene in ovarian tissue, (ii) low expression level of NRDR protein encoded by DHRS4 gene in ovarian tissue, (iii) genotype at Chr7:75245594 of reference genome Sscrofa11.1 is CC;

step 2, breeding F1 generation piglets meeting at least the following two conditions for mating to obtain second generation (F2 generation) piglets: (i) low expression level of mRNA of DHRS4 gene in ovarian tissue, (ii) low expression level of NRDR protein encoded by DHRS4 gene in ovarian tissue, (iii) genotype at Chr7:75245594 of reference genome Sscrofa11.1 is CC;

and 3, breeding F2 piglets meeting at least the following two conditions to obtain third-generation (F3) piglets by mating: (i) low expression level of mRNA of DHRS4 gene in ovarian tissue, (ii) low expression level of NRDR protein encoded by DHRS4 gene in ovarian tissue, (iii) genotype at Chr7:75245594 of reference genome Sscrofa11.1 is CC;

and (3) repeating the step 2 or the step 3 to finally obtain the high-yield-born number breeding pigs with low expression level of DHRS4 gene mRNA in the ovarian tissue, low expression level of NRDR protein coded by DHRS4 gene in the ovarian tissue and CC genotype at Chr7:75245594 of the reference genome Ssicrofa 11.1.

The method follows natural propagation and obtains the breeding pigs with high birth litter size through less manual intervention; meanwhile, the breeding method is a non-transgenic method, the acceptance of the masses is higher, the development of the pig industry can be effectively promoted, and the core competitiveness of the pig breeding industry in China is improved.

The invention also provides a method for identifying or assisting in identifying the litter size of the pigs based on multigroup integrated precise breeding, which at least comprises the following two steps:

step (1), determining the expression quantity of DHRS4 gene mRNA in the pig ovarian tissue, wherein the pig with low mRNA expression quantity has higher birth litter size;

detecting the expression quantity of NRDR protein coded by DHRS4 gene in pig ovarian tissue, wherein the pig with low expression quantity of NRDR protein has higher litter size of birth;

step (3), detecting whether deoxyribonucleotide at a Chr7:75245594 position of a pig reference genome Sscorofa 11.1 is C, or T, or C and T to determine that the genotype of the pig to be detected at the SNP position is CC, TT or TC, or directly determining the genotype by methods such as Sequenom MassArray and the like, and determining the litter size of the pig according to the genotype: pigs with genotype CC have a higher litter size than pigs with genotype TT or TC.

The analysis of the gene level, the transcription level and the protein expression level is integrated, the multigroup science technical means are utilized for analysis, multigroup science information is integrated, the effect of genetic variation on selection and domestication is systematically explained, the multidimensional omics selection of a target group is realized, the accuracy of pig breeding is improved, and the high-stability and high-selectivity pig can be accurately obtained.

Examples

EXAMPLE 1 obtaining molecular markers for SNPs

1. Experimental sample Collection

234 big white pigs from certain pig farms in Shandong were used for SNP typing of the DHRS4 gene. Ear tissue samples were collected one by one, placed in 75% alcohol and stored at-20 ℃ for DNA extraction.

2. SNP typing of DHRS4 gene

2.1 extraction and detection of pig genomic DNA

(1) Cutting a proper amount of pig ear tissues, and putting the cut pig ear tissues into a 1.5mL Axgen tube;

(2) taking a 50mLBD centrifuge tube, uniformly mixing proteinase K with the final concentration of 0.4mg/mL and lysis buffer solution, and adding 0.5mL of lysis solution into a 1.5mL centrifuge tube filled with pig ear tissues for lysis;

(3) the centrifuge tubes were placed in parallel and uniformly on a rocking plate of a constant temperature hybridization oven (the tube caps were tightly sealed to prevent liquid from leaking out). Standing at 55 deg.C for more than 6 hr (it is important to mix the sample completely during digestion process, and the judgment is based on no obvious macroscopic pig ear tissue, and the digested mixture is milky);

(4) after the sample is fully cracked, taking out the centrifuge tube, adding 0.3mL of saturated sodium chloride solution into each tube, reversing and fully mixing for 6-8 times, then placing on ice, and carrying out ice bath for 15 minutes;

(5) after ice-cooling, centrifuge at 12000rpm for 15 minutes at room temperature, carefully and slowly transfer the supernatant to a new 1.5mL Axgen centrifuge tube (care was taken to avoid the pellet from pouring out with the supernatant, keeping the pouring procedure consistent, keeping the amount of supernatant poured out of each tube the same);

(6) adding 0.7mL of isopropanol (the amount of isopropanol added varies with the amount of supernatant poured off, and the two are equal in volume) to each tube, and inverting until flocculent precipitate appears in the solution (if there is no flocculent precipitate, the solution can be left in a refrigerator at-20 ℃ for 2 hours or at 4 ℃ overnight);

(7) centrifugation at 12000rpm for 15 minutes at room temperature removed the supernatant (during which time the tube was carefully observed for white DNA pellet at the bottom of the tube and was not decanted with the supernatant);

(8) 0.5mL of 70% ethanol was added to each tube and inverted gently to wash the precipitated DNA thoroughly;

(9) centrifugation at 10000rmp for 30s, and aspiration of ethanol from the centrifuge tube with a 200. mu.L micropipette to leave the precipitated DNA in the tube (when one is not concerned with aspirating the DNA precipitate from the tube, the tip used in this step may not be replaced when operating between centrifuge tubes);

(10) naturally air-drying the DNA for 10 minutes;

(11) taking 0.1mL of TE buffer solution by a pipette, dissolving the DNA precipitate again, placing the DNA precipitate at 55 ℃ for 2 hours, and shaking for several times to fully dissolve the DNA;

(12) after the DNA is fully dissolved, measuring the extracted concentration (the concentration and OD value of the DNA of a standard sample are needed for genotyping, the concentration of the DNA is 15-20 ng/mu L, the volume is 30 mu L, A260/230 is between 1.5 and 2.3), and detecting the extracted mass (a single visible band) by agarose gel electrophoresis;

(13) the DNA solution was left at 4 ℃ overnight, and 1. mu.L of the DNA solution was subjected to PCR the next day (if the extracted DNA was used within several weeks, it could be stored at 4 ℃ C.; if it was not used for a long time, it was left at-20 ℃ C.).

2.2 genotype determination and quality control of genotype data for SNP chip

DNA samples of 234 white pigs and related information of SNP are submitted to Sequenom MassArray nucleic acid mass spectrometry sequencing by Beijing Conpson organism Limited.

Adding PCR amplification primer (P) with sample DNA as template₁And P₂) And carrying out PCR amplification reaction on the single base extension primer, purifying a reaction product, co-crystallizing the reaction product with a mass spectrum chip, detecting and parting by using a flight mass spectrometry, and detecting the SNP locus of the target gene.

2.3 data collation and analysis

1) Phenotypic data analysis

And performing descriptive statistical analysis on the birth character of the initial birth and the birth character measured value of the multiparous (2-8 times) pig by using SAS9.2 statistical analysis software, wherein the descriptive statistical analysis comprises calculation of the average value, standard deviation, maximum value and minimum value of the characters.

2) Haplotype analysis

The calculation of the genotype and allele frequency of the single nucleotide polymorphism was performed using PopGene 3.2. The GLM process in SAS9.2 software is used for analyzing the association analysis of SNP and characters such as litter size, and the fixed effect model is as follows:

y＝μ+gi+mk+e

y is a phenotypic record of reproductive traits; μ is the total average of the traits; gi is the genotype effect; mk is the production month effect; e is the random error. Data are presented as probability values and mean ± standard deviation, # with P value < 0.05 indicating that the difference is statistically significant, and # with P value < 0.001 indicating that the difference is highly statistically significant.

2.4 analysis of results

The SNP typing results of DHRS4 gene of the white pig were obtained by Sequenom MassArray as described above, and are shown in Table 1.

TABLE 1

The research finds that the DHRS4 gene has an SNP locus rs701332503 which is obviously related to the reproductive traits of sows and is positioned at Chr7:75245594 of a pig reference genome Sscrofa 11.1. The result of the rs701332503 genotype test shows that: the genotype of 37 pigs is CC genotype, the genotype of 69 pigs is TT genotype, and the genotype of 128 pigs is TC genotype. The results of detecting the genotype frequency of the pig DHRS4 gene in the swinery are as follows: the CC genotype frequency is 0.158, the TT genotype frequency is 0.295, the TC genotype frequency is 0.547, the TC genotype frequency is higher than that of CC and TT, and TC allele is a dominant gene.

And (3) carrying out statistical analysis on the genotype and the reproductive traits of the sows by using SAS9.2 software, and carrying out multiple comparison among samples. The results are shown in Table 2.

TABLE 2

As can be seen from Table 2, the difference in parity number between different genotypes at the locus rs701332503 is extremely significant. In the initial production data, the litter size, the pit weight and the average weight average of the TT type and the CT type are slightly higher than those of the CC type, but no statistical significance exists; in the menstruation yield data, the average fetal weight of TT type and CT type is slightly higher than that of CC type, but no statistical significance exists; the litter size and litter weight of the type CC warp is higher than those of the type TT and the type CT, and the difference of the warp litter size has high statistical significance.

Therefore, the SNP locus (rs701332503) has obvious influence on the litter size of the pigs, and the pigs with CC genotypes have higher breeding efficiency in the actual pig breeding.

In summary, the nucleotide of the SNP site (rs701332503) of the pig DHRS4 gene can be determined to determine whether the pig individual is CC genotype or TT and CT genotype, thereby assisting in identifying the born number: the CC genotype pig has higher breeding efficiency.

The CC genotype is the homozygote of the DNA of the rs701332503 locus of the pig which is C.

The CT genotype is that the deoxyribonucleotide of the rs701332503 locus of the pig is a heterozygote of C and T;

the TT genotype is the homozygote of the pig rs701332503 locus deoxyribonucleotide which is T.

Example 2 detection of DHRS4 Gene expression level

1. Experimental sample Collection

The white pig and the Meishan pig for detecting the DHRS4 gene expression are from the pig farm of the animal veterinary institute of Beijing, national academy of agricultural sciences in Wuqing, Tianjin. Collecting ovaries of sows at 180 days and 300 days after birth, storing the samples in liquid nitrogen, and extracting total RNA. Three individuals were collected for each variety as biological replicates. Wherein the Meishan pig is one of the varieties with strongest reproductive capacity and most litter size in the breeds in China, and the litter size of the Meishan pig is 30-40% higher than that of modern European breeds such as white pigs. The influence of the DHRS4 gene expression quantity on the born number can be effectively proved by comparing the DHRS4 gene expression quantities of the Meishan pigs and the white pigs.

2. Detection of DHRS4 Gene expression

2.1 Total RNA extraction from ovarian tissue

Glassware and tweezers used in the RNA extraction process need to be subjected to dry baking for 4 hours at 200 ℃ to inactivate the RNase, an Eppendorf tube and a pipette tip need to use RNase inactivation products, and experimenters need to wear masks and latex gloves in the whole experimental process.

(1) Collecting samples: placing the collected ovarian tissues in a 1.5mL Eppendorf centrifuge tube, and operating according to the RNA extraction instruction;

(2) grinding the tissue on ice by using a grinding pestle, adding 1mL of Trizol lysate, violently shaking for 30s, standing for 10min at room temperature, and fully cracking tissue cells;

(3) adding 0.2mL chloroform (chloroform: Trizol volume ratio is 1:5), vigorously shaking for 15s, standing at room temperature for 10min, centrifuging at 12000g at 4 deg.C for 15min, and repeating the steps until extraction is complete;

(4) sucking the upper aqueous phase (about 450 mu L) into a new Eppendorf tube, adding isopropanol with the same volume, gently mixing, standing on ice for 20min, and centrifuging at 12000g for 10min at 4 ℃;

(5) centrifuging to obtain white colloidal precipitate at the bottom of the tube, discarding the supernatant, washing the precipitate with 1mL of 75% ethanol (prepared with DEPC water), centrifuging at 4 deg.C for 10min at 12000g, discarding ethanol, and air drying on a clean bench for 5-10min until the precipitate is semitransparent;

(6) adding DEPC water to dissolve the RNA precipitate, and determining the amount of the added DEPC water according to the amount of the precipitate;

(7) and measuring the RNA concentration of the sample by using an ultraviolet spectrophotometer.

2.2 Total RNA quality detection method

1 μ L of RNA in the extracted sample was collected, and the quality of RNA was measured by Agilent 2100Bioanalyzer and agarose gel electrophoresis. Taking a sample with the RIN value (RNA integer number) of more than 8 as a library establishing sample. And simultaneously, detecting the integrity of the RNA by adopting 2% agarose gel electrophoresis, and selecting a sample with high integrity for subsequent mRNA quantification.

2.3 reverse transcription of mRNA

Measuring RNA concentration with ultraviolet spectrophotometer, adding 2 μ g RNA into 2 μ L Oligo (dT), adding DEPC water to make up volume to 13 μ L, mixing well, water bathing at 72 deg.C for 5min, standing on ice for 3-5min, centrifuging, adding the following substances, mixing well, water bathing at 42 deg.C for 60min, and storing at-20 deg.C.

TABLE 3 reverse transcription reaction System

2.4 quantitative PCR

The Real-time quantitative PCR reaction was performed according to the kit instructions of TaKaRa SYBR Premix EX Taq (TaKaRa, DRR 041S). Each sample is provided with three replicates, the reaction is carried out in an ABI Prism 7500 real-time fluorescence quantitative PCR system, and beta-actin is an internal reference. The PCR reaction system is shown in Table 4, the reaction conditions are shown in Table 5, and the primer sequences are shown in Table 6.

TABLE 4 RT-qPCR reaction System

TABLE 5 RT-qPCR amplification procedure

TABLE 6 mRNA qPCR primers

2.5 analysis of results

The results of examining the influence of the expression level of DHRS4 gene on the litter size of parity are shown in FIG. 1. FIG. 1 shows the expression difference of DHRS4 gene mRNA level in the ovary of white pig and Meishan pig.

As can be seen from FIG. 1, the expression level of DHRS4mRNA in Meishan pigs with high litter size is significantly lower than that of white pigs, which means that the litter size of the pigs can be identified or assisted to be identified by detecting the mRNA expression of DHRS4 gene in ovarian tissues.

Example 3 detection of an index of expression level of DHRS4 Gene-related protein

1. Tissue sample processing and Total protein quantification

Tissue sample treatment: ovarian tissue was removed from liquid nitrogen and 100. mu.L of RIPA lysate was added to a final concentration of 1mM DTT and PMSF just prior to use. Grinding the tissue by using a grinding pestle, repeatedly freezing and thawing in liquid nitrogen for several times, and fully cracking cells; standing on ice for 30min, then 12000g, centrifuging at 4 deg.C for 30min, and collecting supernatant;

and (3) total protein quantification: standards were prepared as in Table 7 following the BCA protein quantification kit protocol, and 10. mu.L of sample was added after 10-fold dilution. The values were read at 570nm with a microplate reader. Drawing a standard curve by taking A570 as an ordinate and the concentration of the standard substance as an abscissa, calculating the concentration of the protein sample from the standard curve, multiplying the concentration by a dilution factor to calculate the concentration of a sample stock solution, sucking a sample with proper mass, adding A5 xSDS loading buffer solution, and boiling for 5min to denature the protein. And (5) subpackaging and freezing.

TABLE 7 preparation of protein standards

2. SDS-polyacrylamide gel electrophoresis

The glass plate for preparing the SDS-polyacrylamide gel is cleaned, dried and fixed on a frame, separation gel is prepared according to the following table 8 (the volume required by a piece of polyacrylamide gel with the thickness of 1 mm), the separation gel is stood at room temperature until the separation gel is coagulated for about 30 minutes, then concentrated gel is prepared according to the table 8, and the separation gel is inserted into a lane separation comb and stood at room temperature until the separation gel is coagulated for about 40 minutes.

TABLE 8 isolation and concentrate formulation Components

The total protein 50. mu.g/lane was loaded and electrophoresis was continued at 80V for 20min and at 160V for about 45 min.

3. Rotary film

3.1 cutting the gel in which the target protein is positioned according to the pre-dyed protein molecule standard after the electrophoresis is finished, and then soaking the gel in a membrane transfer buffer solution;

3.2 taking out the membrane-transferring electrophoresis tank, carefully placing a stirrer at the bottom, and pouring half of the membrane-transferring buffer solution;

3.3 shearing PVDF membrane, slightly larger than gel, soaking in a little methanol for several seconds in a culture dish, then placing in membrane transferring buffer solution for soaking for 10min, and taking out for use. Taking another two pieces of filter paper and two square sponge pads, and soaking the filter paper and the square sponge pads in a membrane transfer buffer solution for later use;

3.4 taking out the transfer film clamp, opening and horizontally placing, firstly placing a square sponge cushion on the red surface, laying a piece of wet filter paper, carefully placing the wetted transfer paper without trapping air bubbles; after dripping a plurality of drops of buffer solution on the transfer printing paper, spreading a film carefully on the transfer printing paper, then covering a layer of filter paper and another piece of sponge, and finally clamping the whole transfer printing paper;

3.5 clamping the transfer film into a transfer groove which is already provided with a half of transfer buffer solution, and taking note that the side with the PVDF film faces to the positive electrode (red) and the film side faces to the negative electrode (black);

3.6 when the membrane transferring clamp is placed in the membrane transferring groove, placing an ice box, covering a cover, pouring a membrane transferring buffer solution, then placing the membrane transferring buffer solution on a stirrer, opening the stirrer to start stirring, simultaneously connecting a power supply, performing constant-pressure wet conversion for 60min under the voltage of 100V, and transferring the protein to the PVDF membrane;

3.7 the electroporated membranes were stained with ponceau, where clear protein bands were visible, the PVDF membranes were fine cut as indicated by the pre-stained protein molecular standards, and finally washed off ponceau.

4. Antibody incubation, visualization and data analysis (for example, MAP3K8 and GAPDH)

4.1 electroporated PVDF membrane, with 5% skim milk powder or BSA (in 0.05M Tris buffer saline, TBS, pH 7.4) blocking solution at room temperature for 3h incubation;

4.2 adding NRDR antibody diluted 1:2000 and incubating at 4 ℃ for 48 h;

4.3TBST (TBS added with 0.1% tween), 3 times 10min each; adding a goat anti-mouse secondary antibody (GAR-HRP,1:10000) marked by horseradish peroxidase, and incubating for 2h at room temperature; TBST washing, 3 times of 10min each time;

4.4ECL method color development: the Millpore kit was used, following the instructions. The substrate and enzyme were mixed in a 1:1 ratio and left at room temperature for about 10min to return to room temperature. Placing the membrane protein face upwards in a clean vessel, then dripping the luminous reaction liquid on the vessel, placing the membrane in a self-sealing belt after 1-2min, tabletting and exposing; then carrying out development and fixation;

4.5PVDF protein membrane with antibody eluent at room temperature shaking washing 10min, to wash away the binding antibody. Washing with TBST for 3 times and 10min each time to remove eluate;

4.6 adding beta-actin mouse-derived monoclonal antibody diluted by 1:10000, and incubating overnight at 4 ℃;

4.7TBST washing 3 times for 10min each. Adding a secondary antibody GAR-HRP diluted by 1:20000, and incubating for 3h at room temperature;

4.8 washing and ECL color development are the same as above;

4.9 optical Density analysis: the optical density value of the immunolabeling band, i.e., IDV (integrated density value), was analyzed by ImageJ software. GAPDH was used as internal reference. The ratio of the target protein to the internal reference optical density reflects the expression level of the target protein.

The results of examining the influence of the expression level of DHRS4 gene on the litter size of born eggs in ovarian tissues are shown in FIG. 2 and FIG. 3. FIGS. 2 and 3 show the expression difference of the NRDR level of DHRS4 gene protein in the ovary of big white pig and Meishan pig.

As can be seen from fig. 2 and fig. 3, the expression level of the NRDR of the DHRS4 protein in the ovarian tissue of the meishan pigs with high litter size is significantly lower than that of the large white pigs, which means that the litter size of the pigs can be identified or assisted to be identified by detecting the expression of the NRDR of the DHRS4 protein.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Sequence listing

<110> institute of Buddha science and technology

<120> SNP molecular marker related to pig birth litter size, identification and application thereof

<130> 2018

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Claims

1. A molecular marker related to the litter size of swine menstruation, wherein the molecular marker is positioned at Chr7:75245594 of a swine reference genome Sscrofa11.1, the NCBI reference SNP number is rs701332503, and two alleles of the SNP site are C and T.

2. The molecular marker of claim 1, wherein the three genotypes corresponding to the SNP loci are CC, TT and TC genotypes, respectively, and the swine with the CC genotype has higher litter size than the swine with the TT and TC genotypes.

3. The molecular marker of claim 1, wherein the method for detecting the SNP genotyping is performed by a PCR amplification reaction in which an amplification primer P is included₁And P₂The nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

4. The molecular marker of claim 1, wherein the method for detecting the SNP genotyping is performed by a single base extension reaction, wherein the nucleotide sequence of the extended primer in the single base extension reaction is shown as SEQ ID NO. 3.

5. The molecular marker of claim 1, wherein the kit for detecting the SNP site comprises a set of amplification primers P₁And P₂An extension primer, a PCR reaction reagent and a PCR product purification reagent,

the amplification primer P₁And P₂The nucleotide sequences of (A) respectively comprise sequences shown in SEQ ID NO.1 and SEQ ID NO. 2;

the nucleotide sequence of the extension primer comprises a sequence shown in SEQ ID NO. 3.

6. The method for identifying or assisting in identifying the litter size of the pigs is characterized by comprising the steps of detecting whether deoxyribonucleotide at Chr7:75245594 of a pig reference genome Ssicrofa 11.1 is C or T so as to determine the genotype of the pig to be detected at the SNP site is CC, TT or TC, and determining the litter size of the pigs according to the genotype: compared with the pig with the genotype of TT or TC, the pig with the genotype of CC has higher birth cost,

the NCBI reference number of the SNP site at Chr7:75245594 of the pig reference genome Ssicrofa 11.1 is rs701332503,

wherein, the CC genotype is the homozygote of the DNA of the SNP locus of the pig which is C; the TT genotype is the homozygote of the deoxyribonucleotide of the SNP locus which is the T; the TC gene type is a heterozygote of T and C which is the deoxyribonucleotide of the SNP site of the pig.

7. A breeding method of high-yield litter size pigs is characterized in that the method comprises the steps of selecting pigs with CC genotype at Chr7:75245594 of reference genome Sscrofa11.1 as parents for breeding,

wherein the NCBI reference number of the SNP site at Chr7:75245594 of the pig reference genome Sscrofa11.1 is rs 701332503.

8. A breeding method according to claim 7, further comprising determining the litter size of the born product by measuring the expression level of mRNA of DHRS4 gene in pig ovarian tissue;

9. A breeding method according to claim 7, further comprising determining the litter size by measuring the expression level of NRDR protein encoded by DHRS4 gene in pig ovarian tissue;