CN111676293B

CN111676293B - SNP molecular marker related to Qinghai eight-eyebrow pig litter size trait and application thereof

Info

Publication number: CN111676293B
Application number: CN202010414932.6A
Authority: CN
Inventors: 吴国芳; 王磊; 吴森; 马玉红
Original assignee: Qinghai Academy of Animal Science and Veterinary Medicine
Current assignee: Qinghai Academy of Animal Science and Veterinary Medicine
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2023-06-20
Anticipated expiration: 2040-05-15
Also published as: CN111676293A

Abstract

The invention discloses an SNP molecular marker related to the litter size trait of Qinghai eight-eyebrow pigs, and belongs to the technical field of molecular breeding. SNP locus of the SNP molecular marker is positionedDRD1The 1159 th position of the gene, the base of which is one of C, T or G, wherein the nucleotide sequence of the DRD1 gene is shown as SEQ ID NO. 1. The molecular marker and the method can be utilizedDRD1Can be used as a candidate gene, can be utilized in breeding and actual production of the population, and provides theoretical reference for molecular marker assisted selection of Qinghai eight-eyebrow pigs.

Description

SNP molecular marker related to Qinghai eight-eyebrow pig litter size trait and application thereof

Technical Field

The invention belongs to the technical field of molecular breeding, and particularly relates to an SNP molecular marker related to the litter size trait of Qinghai eight-eyebrow pigs and application thereof.

Background

Dopamine receptor 1 (DRD 1, dopamine receptor D1) is a typical G-protein coupled receptor that plays a decisive role in early embryonic development, particularly in the development of neural stem cells. DRD1 is widely expressed in heart, kidney, intestine, muscle, fat, pituitary, hypothalamus, uterus, fallopian tube, etc. tissues of geese. Polymorphisms in the DRD1 region of Columbia (rs 686, rs 1039089) may cause depression. A certain light stimulus can cause the expression level of DRD1 in nerve cells to increase, thereby inhibiting depression behavior. Whereas changes in DRD1 levels in the inner amygdala can cause changes in the vole behavior in grasslands. Polymorphisms in the DRD1 rs4532 region are associated with primary hypertension in asians and africans. In the related research of embryo development of the laying hen through the GWAS research, the SNPs of the DRD1 are possibly related to the egg quality of the laying hen, and the aggressiveness of the laying hen after the DRD1 isogene is knocked out is obviously reduced. The SNP of DRD 1G 123A was associated with the body weight and egg quality of hungarian yellow chickens. SNP at broiler DRD 1C 201T, A, G, A, 255G was significantly correlated with sexual maturation, G1073A and day 300The egg yield and the first nest age are obviously related. SNP of cherry valley duck and Xingyi duck DRD 1T 189C, T A is obviously related to characters such as weight, oblique length, chest depth and the like, and is related to serum total protein, globulin, albumin, white/ball ratio, low density lipoprotein and myochest pH _24h And shear forces are significantly related.

Qinghai eight-eyebrow pigs are used as a plateau group of eight-eyebrow pigs, have the advantages of high litter size and good sows, and few researches on DRD1 and related production traits of pigs, especially Qinghai eight-eyebrow pigs, are reported.

Disclosure of Invention

In order to solve the technical problems, the inventor uses modern biotechnology to carry out multidirectional functional analysis on the porcine DRD1 gene, detects SNPs of all exon segments of the Qinghai eight-eyebrow porcine DRD1, carries out correlation analysis on the SNPs and related production and reproduction traits, and can provide reference for molecular auxiliary breeding work of the Qinghai eight-eyebrow porcine in the aspect that 1 SNP is accidentally found to be related to the litter size trait in the exon region of the DRD1 gene, thereby completing the invention.

The first aspect of the invention provides an SNP molecular marker related to the litter size trait of Qinghai eight-eyebrow pigs, wherein the SNP locus of the SNP molecular marker is positioned at 1159 th site of a DRD1 gene, and the base is one of C, T or G, and the nucleotide sequence of the DRD1 gene is shown as SEQ ID NO. 1.

The DRD1 gene is positioned on the 16 th chromosome of the pig, the total length of the DRD1 gene is 4168bp, the DRD1 gene comprises two exon regions, and the reference sequence is GeneBank No. NC_010458.4 (SEQ ID NO. 1). Its protein (GeneBank No.: NP-001116580.1) consists of 446 amino acids, with functional domains such as glycosylation and multiple transmembrane structures.

In a second aspect, the invention provides the use of a reagent for detecting the SNP molecular marker according to the first aspect of the invention in the preparation of a kit for identifying the litter size trait of Qinghai eight-eyebrow pigs.

In a third aspect, the invention provides a kit for identifying the litter size trait of Qinghai eight-eyebrow pigs, comprising reagents capable of detecting the SNP molecular markers described in the first aspect of the invention.

In some embodiments of the invention, the agent is a primer combination capable of amplifying the SNP molecular markers.

In some embodiments of the invention, the primer combination consists of an upstream primer having the nucleotide sequence set forth in SEQ ID NO.2 and a downstream primer having the nucleotide sequence set forth in SEQ ID NO. 3.

The third aspect of the invention provides a method for breeding Qinghai eight-eyebrow pigs with more farrowing number, which comprises the following steps:

(1) Obtaining genomic DNA of Qinghai eight-eyebrow pigs;

(2) Detecting the genotype of the SNP molecular marker of claim 1 in the genome DNA, selecting CT, TT, CG or GG genotype Qinghai eight-eyebrow pigs, and eliminating other genotypes Qinghai eight-eyebrow pigs.

In some embodiments of the invention, the genotype of the SNP molecular marker is detected in step (2) using a primer combination.

In some preferred embodiments of the invention, CT or TT genotype green sea eight eyebrow pigs are selected and other genotypes green sea eight eyebrow pigs are eliminated.

In some more preferred embodiments of the invention, the TT genotype Qinghai eight-eyebrow pig is selected and the other genotype Qinghai eight-eyebrow pig is eliminated.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following beneficial technical effects:

the DRD1 gene has polymorphism in Qinghai eight-eyebrow pigs, and C1159T is the main mutant type. C1159T was significantly associated with the litter size of Qinghai eight-eyebrow pigs, with TT-type populations exhibiting the highest litter size, averaging 10.2 heads.

The molecular marker and the method can utilize DRD1 as a candidate gene, can be utilized in the breeding and actual production of the population, and provide theoretical reference for the molecular marker auxiliary selection of Qinghai eight-eyebrow pigs.

Drawings

Figure 1 shows a primary structural alignment of pigs and their closely related species DRD 1.

Figure 2 shows a conservative motif alignment in the secondary structure of DRD1 of pigs and their closely related species.

FIG. 3 shows an alignment of conserved domains in the tertiary structure of DRD1 in pigs and closely related species.

FIG. 4 shows the porcine DRD1 protein evolution analysis.

Fig. 5 shows the pig DRD1 protein direct interaction protein Top10.

FIG. 6 shows the main functional enrichment of porcine DRD1 protein and direct interaction protein (Top 50).

FIG. 7 shows a map of the SNP locus detection of the DRD1 exon of Qinghai eight-eyebrow pig.

Figure 8 shows parity for each genotype of Type 1.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1

1 materials and methods

1.1 sample collection

333 healthy eight-eyebrow pig sows used in the embodiment come from Qinghai province mutually-assisted eight-eyebrow pig stock breeding farms, and the experimental pig groups have the advantages of consistent breeding background, close weight, good health condition, no relationship within three generations and more than 2 times of parity. Ear tissues were collected, and DNA was extracted using a tengen tissue DNA kit (langen, beijing) and stored at-20 ℃ for use.

1.2 determination of growth and propagation Properties

The growth traits of Qinghai eight-eyebrow pigs were determined and recorded: body length (Body length), body height (Chest circumference), hip circumference (Hip circumference), backfat thickness (Thickness of backfat), backfat thickness measured by a veterinary backfat instrument (B ultrasonic), and other properties measured and recorded by flexible ruler and stick, and data measured 3 times; reproduction traits: number of parity (number), average parity of parity via sow parity statistics; the number of teats (Teat number) was recorded and counted as the average number of teats on both sides.

1.3DRD1 biometric information analysis

The mammalian sequences of pigs and related common patterns were searched at NCBI, specifically: human (NP 000785.1,Homo sapiens), mouse (NP 034206.1,Mus musculus), rat (NP 036678.3,Rattus norvegicus), cow (NP 776467.1,Bos taurus), pig (NP 001116580.1,Sus scrofa), zebra fish (NP 001129448.1,Danio rerio). Multiple sequence comparisons were performed using the mule method using MEGA X software (version 10.1.7); phylogenetic trees were constructed using poisson model in a contiguous method, species evolutionary relationships were analyzed, and data visualization was performed using CLC Sequence view (version 8.0). Functional motif structures in the secondary structure of the multi-species DRD1 protein were detected by MEME wait (version 5.1.1) on-line software (http:// me-wait. Org /), and the results were visualized as TBtools (version 0.6735). Mining interaction of pig DRD1 and direct interaction protein by using STRING database (version 11.0) (https:// STRING-db. Org /), mining main functions of DRD1, and visualizing a STRING network interaction result by using Cytoscape (version 3.7.2); the functional enrichment results were sorted with dplyr (version 0.8.5) of R (version 3.6.3), visualized with cowplot (version 1.0.0) and ggplot2 (version 3.3.0); the function enrichment items are fewer than 20 direct outputs, and more than 20 only output Top20. Protein conserved domain detection (https:// www.ncbi.nlm.nih.gov/CDD) was performed using the NCBI CDD (Conserved Domains) database, and the results were visualized as TBtools.

1.4 PCR amplification and SNP detection of Qinghai eight-eyebrow pig DRD1 target gene

The Qinghai eight eyebrow pig DRD1 gene sequence is as follows (SEQ ID NO. 1):

AGTGCCAGCTCGTCTCTCCGCCTCGTGTTCCCACGGGCTCTTGGGTGATAGCGGGCAGTTTCGCGGGCAGGCGCAGGGCTGTTCCTCGAGGGACCAGAGACCACTCGAGGGGCGCCCGGGTGCTCGGCTCCCGGAGAAAGGAAGCAAAGAAACTGCCCGAGTGACTACTACAGGAGGTGTCCTCAGTCAAGGAGGCGCCTAGCGCCCGAAAGCCCTTTTCCCGGGTCTCGTCCATTTTGAAGCATCTCTAACCTTCTGAGGCAGTGGCGCAGCGCCTGCTCTCCGTGGACTTGGCCCAGGATCCTTTTTCCGAACCCGCCCCAGCGAATTTTGCGCATCGGGTGGGGAGCAGAGCCCCGGCTGCGCGCGCAGGGCAGGACTCAGGCGCGCCTCCCTCCGTTGTGCAGCTGCGGCGGGCGCTTGGGGAATCCTGTTCTCGGAGCTCCAGGAGCATTGAGAGAGACCACCTCAAGGCAAGGTAGGTGGCTCTCCCGGAGTTGGATTGGGCTTGGCAAGTAAAGATGCGATCTGCTCAAATCTTGGCAAACGTCCTCAGCTCTCTCTTTGCTTTCTAGGGCTTTCGGGGAGCTGCTGCGGAGTCAGGGGCTTGGAGGTGCGAGGCATGTATTTTCAGCTGCGCCTCAGAAAGGGAGAATTCTCTGTCACCACCCGAGAGGCAACAGCCCGTAAATGTGACTACAATTGACTAGCTCGGCAGAGGCCTGGGAGTCTCTGAACTGACAGCTTAGAATATGCTAAAAAGCCAGTGCTTTCCATGGGGCATTGAAGGGCCATCTGGTCCCCGTAACAGTGACCTGAAGCAAAGGAGTCAGAAGACAGTTGTAGAGAGCAAGAGGGACCTTCCGAGGGGCTGTCTTGGACGGCCCAGGGCAGGCTCCTGCCGCCCAGTTGTTGTGGCCTTGTGATGGCACGCTGGTGAGGTCCTCCACCCAGGGGAGCAAGTGGCGCTGAGCCAGGGACCGCCTGGCCAGCCAGGACTCCTGCAGCTCTGATCGACCCCTAATCCCCGCCTAGGAACTTGAGGGGTGTCAGAACCCCCTCTGGGCTCTCCCTCAGGAAGATGAGGACGCTGAACACCTCCACCATGGACGGCACCGGGCTGGTGGTGGAGAGGGACTTCTCCTTCCGCATCCTCACCGCCTGTTTCCTGTCGCTGCTCATCCTGTCCACACTCCTGGGGAATACGCTGGTCTGTGCCGCCGTCATCAGGTTCCGACACCTGCGTTCCAAGGTGACCAACTTCTTCGTCATCTCCTTAGCCGTATCGGATCTCTTGGTGGCTGTCTTGGTCATGCCCTGGAAAGCGGTGGCTGAGATCGCTGGCTTCTGGCCCTTTGGGTCCTTCTGTAACATCTGGGTGGCCTTTGACATCATGTGCTCTACCGCGTCCATCCTCAACCTCTGTGTGATCAGCGTGGACAGGTACTGGGCCATCTCTAGCCCCTTCCGGTATGAGAGGAAGATGACCCCCAAGGCAGCCTTCATTCTGATCAGCGTGGCATGGACTCTGTCCGTTCTCATCTCCTTTATCCCAGTGCAGCTCAGCTGGCACAAGGCAAAACCCACGAGCCCCTCCGATGGGAATGTCACTTCCCTGGGCAAGACCACCCACAACTGTGACTCCAGCTTGAGCAGGACCTATGCCATTTCATCCTCCCTAATCAGCTTTTACATCCCCGTGGCCATCATGATTGTCACCTACACCAGGATCTACAGGATCGCCCAGAAACAAATACGGCGCATCTCGGCCTTGGAGAGGGCAGCGGTCCACGCCAAGAATTGCCAGACCACTGCAGGTAATGGCAACCCTGCGGAGTGTTCTCAACCAGAAAGCTCCTTTAAGATGTCCTTCAAAAGAGAGACTAAAGTCCTGAAGACGCTGTCCGTGATCATGGGGGTGTTTGTGTGCTGCTGGCTCCCCTTCTTCATCTTGAACTGCATGGTGCCCTTCTGTGGGTCTGGGGAGACCAAGCCCTTCTGCATTGATTCCATCACCTTTGACGTGTTTGTGTGGTTTGGGTGGGCTAATTCCTCCTTGAACCCCATCATCTATGCCTTTAATGCTGATTTTCGGAAGGCATTTTCCACCCTCTTAGGATGCTACCGACTCTGCCCGACGTCAACTAATGCCATAGAGACGGTGAGCATCAATAACAATGGGGCCGTGGTGTTTTCCAGCCATCACGAGCCGCGAGGCTCCATCTCCAAGGACTGCAATCTGGTGTATCTGATCCCCCATGCTGTGGGCTCCTCTGAGGACCTGAAGAAGGAAGAGGCAGGTGGCATAGCCAAGCCCTTGGAGAAGCTGTCCCCAGCCCTGTCTGTCATTTTGGATTATGACACTGATGTCTCTCTAGAGAAGATCCAGCCCATCACACAAAATGGACAGCACCCGACCTGAACTCCAAGGTGAATCTTAACAGACCCACTCATCCCAAAAGCTGGAGGAGATTTCTCTGGGGCTTGTTGTGAAGAAACTGAGGCGTGATGAGACCCTGAGCTGTCAGGCGAGCCCTCCTCTGCTGCTTTCCCTCCAACCCACCACTAACCACATTTTAAAATACGTTCCAATGTGTTTTCTGTGTTGTTCATAGTGAATCAGAGGGACACATGTGAGGCGATCATTCATAAGGGACGTGTCTTTGGCTCCAAAATTATTTTTAGAAACTGATTCTTATCTTAGGACTTTAAAAAATAGGGCACAGAAGCAAGAAATGAAGAGCTTCATTTAAAAATTAGATTTTTCCGGGAAGGAAATGAGAAGGGTTGAGTTTGCTGTGTACAAACAGGTGCTAACACTGGTCCAAGCAAAGTTTTCAGATTGTAAAGGTAGGTGCATGCCTTCATAAATTATTCCTAAAAAAGAATTGAGCCTTACAATAGGAATGGGATTTTTTTTTCCAGTGTTGATGCTTTGTTGGTATTGGTTTTATTTATTTATTGTATTATATGGATATTTAAAATTTATTATAATAAATCTATATTTATCATATTTAATAGGATAAATGAATGAGTTTTCTGAGATCTTACAATAGCATTTTTTGTCCATTGAACTAGCACTTTATCAGCCAGTGAAACAAATACACAGACTCTCTGAGTTTCTAAATGCTCATATAAAACTTCCAGAAATACAGCAAAGACTAATAGAAACTGAAGTTGTGAGGATTCCTTAAAATTCATTGGCAAGAATAAATTCGAGGTGAGAATTCACAAACGCTCAGATTGTTTTTTTTTTTCCTCCTGAAAAGATTTAGAAAGATTTTTTAAAAAGCATAGCTCCTCCTGTGTTCAGATTTTTTTAAGTGACGAAGACTTTTGCCTGAGAATGAGTTACAGTTCTGTAAATATCTGAAATAAAAAACAGCTTAAGCGTCCAGCTTGAAATTTACGACCTTTGGTGGTAATAAAAAGTATTTGCCACTTTGTATTTATGTAAAATAACTGGCCCTCTCTGTCTTTTTTCATTTCCTGTGTCAGATAGCTTTTCTGAACCAAATAAATGGCTGTCCTGGTTAGATTTGTGGGAAGACAGTGAGTTTCCTTAACTCATGTGTCACAACAGGTTCACCAGTGGACCAAGGTCAGATCTTACACCACCATCTTACCAGGTCAAACCAAGCTTTTCAGTGGGGCTACTTTTCGTAGTGCTTTAATCTGAACTGAGAATTTTTTTTTTTTTAAGTCTAAATGTATTCTAACAGATAGTGCCTCATTATCTTCCTCAAGTAAGACACTTCTGTTGGTGGAAAAAAACAGGCGACCCTTTCCTTTCTCTTTGGGTATATTAAGCATCCCAAAGCCATCAGCATCTCTTTTGACAAATGCTAGCCCTTTCTCTGTGCTTTGGAATCAAGTTCCTGTGTCATCATCTGGGCTGTAAAAAGTATCATAAATCTCCATTGCCAGCTAGTAATTCATGTGGGGCATTTCAAGAGAATTTTTTTGGAAATGTTTACAAAGTATTCTTGTGGGTGAGCAATTGACTTAACATTTACTTGAAATGGGTAAGGAGAAGGATACCTACTGCAAAGGATCTCCTGCATCAGGTCTGTGTTATTTATGCATTGTGAATGTTTTCTTCATTTTATTGCCTGTATGCTTTCTTACATATAATAAAATTATTTTGTGAACTCAGATCAACCCTAAA

PCR amplification primers were designed for all exon regions (NM-001123108.1) of the porcine DRD1 gene, and the primer sequences are shown in Table 1.

30 DNA samples were randomly selected, and an equal amount of DNA was aspirated from each sample to construct a DNA mix. PCR amplification was performed using the DNA mixed sample as a template, and the PCR product was electrophoretically detected using 1.0% agarose gel:

20.0 mu L of PCR reaction system, comprising: dNTPs containing nucleic acid dye, taq DNA polymerase, 10 xMIX 10.0. Mu.L, ddH ₂ O7.4. Mu.L, 0.8. Mu.L of each of the upstream and downstream primers (10 pmol/. Mu.L), and 1.0. Mu.L of the template DNA (50 ng/. Mu.L).

Cycling scheme: 95℃for 5min (pre-denaturation); 94℃for 30s (denaturation); 55.0deg.C for 30s (annealing, specific annealing temperatures are shown in Table 1); 1min (extension) at 72 ℃; and (3) preserving at 72 ℃ for 10min (final extension) and 4 ℃.

Products meeting the sequencing standards were sequenced by Shanghai Megaku Biotechnology services Co. Sequencing results sequence alignment and analysis were performed by DNA STAR software (version 7.1) software. Amplifying, sequencing and typing detection are carried out on SNP fragments existing in 333 samples one by one according to the detection result.

TABLE 1 DRD1 primers and PCR amplification information

1.5 statistical analysis

On the basis of identifying SNP, the growth traits and the reproduction traits of the eight-eyebrow pigs are related, and SPSS (version 26) software is adopted to count the related results, and a general linear model is adopted: phenotype value = population mean + marker genotype effect + age effect + fetal time effect + random error.

2 results and analysis

2.1 porcine DRD1 protein and sequence characteristics of related species thereof

The multi-sequence comparison result shows that a large number of conserved fragments exist in the primary structure of the pig DRD1 protein and DRD1 of animals such as human, rat, mouse, cow, zebra fish and the like (figure 1); there are a number of significant conserved motif structures on the secondary structure (fig. 2); there is one identical PHA03087 conserved functional domain in tertiary structure (FIG. 3). DRD1 is relatively structurally conserved in pigs and their closely related species, so DRD1 functions may be similar in each species.

2.2 pig DRD1 protein genetic evolutionary analysis

The results of FIG. 4 show that porcine DRD1 protein has the closest evolutionary relationship to bovine, inferior to murine, and furthest away from zebra fish in terms of species genetic evolution.

2.3 pig DRD1 protein and direct interaction protein relation thereof

The results of the STRING network interaction show that the pig DRD1 protein has strong interaction and regulation effects with 10 proteins such as CRH, NPS, ADCY, ADCY9 and the like (figure 5). From FIG. 6, it can be seen that the porcine DRD1 protein is mainly related to activities such as circadian rhythm, chronic morphine intoxication, relaxin signaling pathway, gamma-aminobutyric acid synapse, neuroactive ligand-receptor interaction, cholinergic synapse, glutamatergic synapse, adipocyte factor signaling pathway, retrograde endogenous cannabinoid signaling, chemokine signaling pathway, cyclic adenosine monophosphate signaling pathway, etc. through KEGG enrichment. Through GO enrichment, the porcine DRD1 protein plays a role in participating in biological processes (BP, biological_process), mainly in activities such as G protein coupled receptor signaling pathway, adenylate cyclase activating G protein coupled receptor signaling pathway, cell signaling, signal transduction, regulation of cell processes, biomass regulation, molecular function regulation, regulation of signal receptor activity, signal transduction regulation, insulin secretion and the like; in terms of Molecular Function (MF), DRD1 plays a major role in activities such as G-protein coupled receptor activity, hormonal activity, neuropeptide hormonal activity, molecular function modulators, protein binding, adhesives, peptide binding, G-protein coupled peptide receptor activity, enzyme binding, etc.; in terms of participation in cellular_component (CC), DRD1 plays a role mainly in activities such as extracellular region, plasma membrane, components of plasma membrane, and cellular components.

2.4 Qinghai eight eyebrow pig DRD1 gene exon region SNP detection

As shown in FIG. 7, 1 SNP was detected in the exon region of the DRD1 gene. Interestingly, there were two mutation types at 1159 of the Qinghai eight eyebrow pig DRD1 gene, labeled Type1 (C > T) and Type2 (C > G), respectively.

2.5 Qinghai eight eyebrow pig DRD1 Gene polymorphism analysis

As can be seen from Table 2, in the DRD1 gene, there are 3 genotypes CC, CT, TT and CC, CG and GG of Type1 and Type2, and the genotype frequencies are 53.0%, 36.7%, 10.3% and 80.6%, 14.2% and 5.2%, respectively; C. t two alleles were 71.4%, 28.6% and 87.7%, 12.3% frequency, respectively; the first mutant C > T is tested by Hardy-Wenberg equilibrium chi-square, polymorphic loci are in an equilibrium state, population genetics are not subjected to manual intervention and genetic drift through long-term evolution and selection, and the selection pressure is low; the observed heterozygosity of Ho is basically different from the expected heterozygosity of He, and the observed heterozygosity of Ho is in low heterozygosity, and the influence of mutation in SNP is moderate; effective allele factors Ne of 1.691, 1.275, respectively, i.e., 1.691, 1.275 alleles, respectively, are required to produce the same homozygous rate for the mutation at one locus under ideal populations; the mutation site PIC value is 0.25< PIC=0.325 <0.5, belongs to medium polymorphism information content, and has certain genetic potential, large mutation potential and large breeding potential. The second mutant C > G has fewer mutant individuals, PIC=0.192 <0.25, belongs to low polymorphism, has small mutation degree and has large selection pressure.

TABLE 2 genotype and mutation frequency distribution of DRD1 in eight-eyebrow pigs

χ ² _0.05 ＝5.991，HWE：Hardy-Weinberg Equilibrium。

2.6 analysis of Qinghai eight-eyebrow pig DRD1 gene SNP related production trait and propagation trait

As can be seen from Table 3, among the two mutants of DRD1 gene, since there are fewer individuals with Type2 mutation, we have mainly explored Type1 mutation. Type1 mutations were significantly correlated with litter size (P < 0.05), with mutant homozygous litter size up to 10.2 heads (fig. 8); the mutation homozygous TT parity number is obviously higher than that of wild homozygous CC and heterozygous CT (P < 0.05), DRD1 can be considered as a candidate gene related to the litter-size trait of the eight-eyebrow pigs in subsequent molecular marker assisted breeding, and T1159T type is the main genotype type for breeding. The mutation has no obvious relation with growth characters such as body size of eight-eyebrow pigs (P is more than 0.05).

TABLE 3 association analysis of SNP loci and traits of DRD1 genes

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Qinghai province academy of veterinary sciences of livestock and veterinary medicine

<120> SNP molecular marker related to Qinghai eight-eyebrow pig litter size trait and application thereof

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agtgccagct cgtctctccg cctcgtgttc ccacgggctc ttgggtgata gcgggcagtt 60

tcgcgggcag gcgcagggct gttcctcgag ggaccagaga ccactcgagg ggcgcccggg 120

tgctcggctc ccggagaaag gaagcaaaga aactgcccga gtgactacta caggaggtgt 180

cctcagtcaa ggaggcgcct agcgcccgaa agcccttttc ccgggtctcg tccattttga 240

agcatctcta accttctgag gcagtggcgc agcgcctgct ctccgtggac ttggcccagg 300

atcctttttc cgaacccgcc ccagcgaatt ttgcgcatcg ggtggggagc agagccccgg 360

ctgcgcgcgc agggcaggac tcaggcgcgc ctccctccgt tgtgcagctg cggcgggcgc 420

ttggggaatc ctgttctcgg agctccagga gcattgagag agaccacctc aaggcaaggt 480

aggtggctct cccggagttg gattgggctt ggcaagtaaa gatgcgatct gctcaaatct 540

tggcaaacgt cctcagctct ctctttgctt tctagggctt tcggggagct gctgcggagt 600

caggggcttg gaggtgcgag gcatgtattt tcagctgcgc ctcagaaagg gagaattctc 660

tgtcaccacc cgagaggcaa cagcccgtaa atgtgactac aattgactag ctcggcagag 720

gcctgggagt ctctgaactg acagcttaga atatgctaaa aagccagtgc tttccatggg 780

gcattgaagg gccatctggt ccccgtaaca gtgacctgaa gcaaaggagt cagaagacag 840

ttgtagagag caagagggac cttccgaggg gctgtcttgg acggcccagg gcaggctcct 900

gccgcccagt tgttgtggcc ttgtgatggc acgctggtga ggtcctccac ccaggggagc 960

aagtggcgct gagccaggga ccgcctggcc agccaggact cctgcagctc tgatcgaccc 1020

ctaatccccg cctaggaact tgaggggtgt cagaaccccc tctgggctct ccctcaggaa 1080

gatgaggacg ctgaacacct ccaccatgga cggcaccggg ctggtggtgg agagggactt 1140

ctccttccgc atcctcaccg cctgtttcct gtcgctgctc atcctgtcca cactcctggg 1200

gaatacgctg gtctgtgccg ccgtcatcag gttccgacac ctgcgttcca aggtgaccaa 1260

cttcttcgtc atctccttag ccgtatcgga tctcttggtg gctgtcttgg tcatgccctg 1320

gaaagcggtg gctgagatcg ctggcttctg gccctttggg tccttctgta acatctgggt 1380

ggcctttgac atcatgtgct ctaccgcgtc catcctcaac ctctgtgtga tcagcgtgga 1440

caggtactgg gccatctcta gccccttccg gtatgagagg aagatgaccc ccaaggcagc 1500

cttcattctg atcagcgtgg catggactct gtccgttctc atctccttta tcccagtgca 1560

gctcagctgg cacaaggcaa aacccacgag cccctccgat gggaatgtca cttccctggg 1620

caagaccacc cacaactgtg actccagctt gagcaggacc tatgccattt catcctccct 1680

aatcagcttt tacatccccg tggccatcat gattgtcacc tacaccagga tctacaggat 1740

cgcccagaaa caaatacggc gcatctcggc cttggagagg gcagcggtcc acgccaagaa 1800

ttgccagacc actgcaggta atggcaaccc tgcggagtgt tctcaaccag aaagctcctt 1860

taagatgtcc ttcaaaagag agactaaagt cctgaagacg ctgtccgtga tcatgggggt 1920

gtttgtgtgc tgctggctcc ccttcttcat cttgaactgc atggtgccct tctgtgggtc 1980

tggggagacc aagcccttct gcattgattc catcaccttt gacgtgtttg tgtggtttgg 2040

gtgggctaat tcctccttga accccatcat ctatgccttt aatgctgatt ttcggaaggc 2100

attttccacc ctcttaggat gctaccgact ctgcccgacg tcaactaatg ccatagagac 2160

ggtgagcatc aataacaatg gggccgtggt gttttccagc catcacgagc cgcgaggctc 2220

catctccaag gactgcaatc tggtgtatct gatcccccat gctgtgggct cctctgagga 2280

cctgaagaag gaagaggcag gtggcatagc caagcccttg gagaagctgt ccccagccct 2340

gtctgtcatt ttggattatg acactgatgt ctctctagag aagatccagc ccatcacaca 2400

aaatggacag cacccgacct gaactccaag gtgaatctta acagacccac tcatcccaaa 2460

agctggagga gatttctctg gggcttgttg tgaagaaact gaggcgtgat gagaccctga 2520

gctgtcaggc gagccctcct ctgctgcttt ccctccaacc caccactaac cacattttaa 2580

aatacgttcc aatgtgtttt ctgtgttgtt catagtgaat cagagggaca catgtgaggc 2640

gatcattcat aagggacgtg tctttggctc caaaattatt tttagaaact gattcttatc 2700

ttaggacttt aaaaaatagg gcacagaagc aagaaatgaa gagcttcatt taaaaattag 2760

atttttccgg gaaggaaatg agaagggttg agtttgctgt gtacaaacag gtgctaacac 2820

tggtccaagc aaagttttca gattgtaaag gtaggtgcat gccttcataa attattccta 2880

aaaaagaatt gagccttaca ataggaatgg gatttttttt tccagtgttg atgctttgtt 2940

ggtattggtt ttatttattt attgtattat atggatattt aaaatttatt ataataaatc 3000

tatatttatc atatttaata ggataaatga atgagttttc tgagatctta caatagcatt 3060

ttttgtccat tgaactagca ctttatcagc cagtgaaaca aatacacaga ctctctgagt 3120

ttctaaatgc tcatataaaa cttccagaaa tacagcaaag actaatagaa actgaagttg 3180

tgaggattcc ttaaaattca ttggcaagaa taaattcgag gtgagaattc acaaacgctc 3240

agattgtttt tttttttcct cctgaaaaga tttagaaaga ttttttaaaa agcatagctc 3300

ctcctgtgtt cagatttttt taagtgacga agacttttgc ctgagaatga gttacagttc 3360

tgtaaatatc tgaaataaaa aacagcttaa gcgtccagct tgaaatttac gacctttggt 3420

ggtaataaaa agtatttgcc actttgtatt tatgtaaaat aactggccct ctctgtcttt 3480

tttcatttcc tgtgtcagat agcttttctg aaccaaataa atggctgtcc tggttagatt 3540

tgtgggaaga cagtgagttt ccttaactca tgtgtcacaa caggttcacc agtggaccaa 3600

ggtcagatct tacaccacca tcttaccagg tcaaaccaag cttttcagtg gggctacttt 3660

tcgtagtgct ttaatctgaa ctgagaattt tttttttttt aagtctaaat gtattctaac 3720

agatagtgcc tcattatctt cctcaagtaa gacacttctg ttggtggaaa aaaacaggcg 3780

accctttcct ttctctttgg gtatattaag catcccaaag ccatcagcat ctcttttgac 3840

aaatgctagc cctttctctg tgctttggaa tcaagttcct gtgtcatcat ctgggctgta 3900

aaaagtatca taaatctcca ttgccagcta gtaattcatg tggggcattt caagagaatt 3960

tttttggaaa tgtttacaaa gtattcttgt gggtgagcaa ttgacttaac atttacttga 4020

aatgggtaag gagaaggata cctactgcaa aggatctcct gcatcaggtc tgtgttattt 4080

atgcattgtg aatgttttct tcattttatt gcctgtatgc tttcttacat ataataaaat 4140

tattttgtga actcagatca accctaaa 4168

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

actcaggctt agttgagggt c 21

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gctgaaaata catgcctcgc a 21

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctctcccgga gttggattgg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gtgtcggaac ctgatgacgg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atccccgcct aggaacttga 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gatcacggac agcgtcttca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tccagcttga gcaggaccta 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

acagctcagg gtctcatcac 20

Claims

1. The method for breeding Qinghai eight-eyebrow pigs with more farrowing numbers is characterized by comprising the following steps:

(1) Obtaining genomic DNA of Qinghai eight-eyebrow pigs;

(2) Detecting the genotype of an SNP molecular marker in the genome DNA, wherein the SNP locus of the SNP molecular marker is positioned at 1159 th site of a DRD1 gene, and the base is one of C, T or G, and the nucleotide sequence of the DRD1 gene is shown as SEQ ID NO. 1;

CT or TT genotype Qinghai eight-eyebrow pigs are selected, and other genotype Qinghai eight-eyebrow pigs are eliminated.

2. The method according to claim 1, wherein the genotype of the SNP molecular markers is detected in step (2) using a primer combination.

3. The method of claim 2, wherein the primer combination consists of an upstream primer shown in SEQ ID No.2 and a downstream primer shown in SEQ ID No. 3.

4. The method of claim 1, wherein the TT genotype green sea eight eyebrow pigs are selected and the other genotypes green sea eight eyebrow pigs are eliminated.