CN107267627A

CN107267627A - The preparation and application of the Six1 gene molecule marker related to pig production character

Info

Publication number: CN107267627A
Application number: CN201710565071.XA
Authority: CN
Inventors: 吴望军; 刘红林; 刘凯清; 李伯江; 董超; 张增凯
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2017-10-20
Anticipated expiration: 2037-07-12
Also published as: CN107267627B

Abstract

The invention belongs to pig marker assisted selection (MAS) technical field, the preparation and application of the Six1 gene molecule marker related to pig production character are disclosed.The molecular labeling is selected from least one of (1)~(2)：(1) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 2, in sequence table SEQ ID NO：There is 1 A11 G11 mutation at the 11bp of sequence shown in 2, cause HindII RFLP polymorphisms；(2) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 13, in sequence table SEQ ID NO：There is 1 (AC) n microsatellites variation at the 11bp of sequence shown in 13, n represents (AC) repeat number.By population data association analysis, promoter activity, and gene expression analysis, it was confirmed that two molecular labelings are with pig production character in significantly correlated.The preparation method and application for the molecular labeling that the present invention is set up can be used for the molecule seed selection of pig associated production character.

Description

The preparation and application of the Six1 gene molecule marker related to pig production character

Technical field

The invention belongs to pig marker assisted selection (MAS) technical field, it is mainly concerned with 2 kinds and can be used for pig production character mark Remember the preparation and application of the molecular labeling of assisted Selection.One of molecular labeling is located at pig Six1 gene First Introns In A/G variant sites at 1595bp, and another molecular labeling is located at Six1 gene promoter areas (- 1029bp--1100 Area) (AC) n microsatellite variant sites on.

Background technology

China is the first pork producing country, and pork yield is maintained at more than 5.0 thousand ten thousand tons every year in recent years, accounts for whole meat 63% or so of yield, pork is the main body of our people's consumption of meat.Since reform and opening-up, the pork of this local products of China is much The demand of domestic market can not be met, in order to solve the problem of supply of pork consumption market is not enough, China introduces substantial amounts of west Square bacon hogs kind, and gradually form production Duroc × length it is white × production models of great Bai three way cross market pigs.However, with A large amount of introductions of Modern China, in the market frequently occurs various types of porks inferior, be mainly manifested in yellowish pink turn white, meat Turn sour.The appearance of these meat of poor qualities brings huge economic loss to consumption market, while mouth when also having a strong impact on edible Sense.Therefore, heredity is carried out to pig variety important economical trait, particularly Meat Quality from source using genetic improvement technological means Improvement, so that the high quality pork tool for producing high-quality is of great significance.

It is past in decades in, traditional breeding method system achieves length on the genetic improvement of middle high heritability character The property such as the progress of foot, such as speed of growth, feed efficiency, carcass lean meat percentage, but meat, breeding for low genetic force Shape, when carrying out hereditary and selection using traditional breeding method system, not only cost is high, and acquired genetic progress is also very slow. With the fast development in the fields such as genomics, molecular biology, the foundation of molecular breeding technology system can be the low genetic force of pig The genetic improvement of character provides new effective way.Relatively conventional traditional breeding method, molecular marker assisted selection, which has, to be permitted Many obvious advantages, it is not by the age, and the limitation of sex etc. can carry out early stage seed selection, shorten the generation inteval, improve selection Accuracy, improves the intensity of selection, reduces the cost of seed selection, accelerates the progress of genetic improvement.At present, halothane (RYR1) CDNA 1843 sites, the 200th codon site of the subunit genes (PRKAG3) of adenosine monophosphate activated protein kinase γ 3 is equal It has been successfully applied to the molecule seed selection of high-quality boar.Nevertheless, being presently available for pig important economical trait molecular labeling auxiliary The mark of seed selection cans be counted on one's fingers, the major gene resistance of identification influence pig important economical trait, develops corresponding molecular labeling and is applied to The seed selection of high-quality boar, the cultivation to accelerating China high-quality boar, the competitiveness tool of lifting China boar industry in the international market There is important facilitation.

Six1 belongs to Six pa-ncreatic and duodenal homeobox1 family members, is a very important growth adjustment factor, in muscle Grow, special important function is particularly played in the transformation of muscle fiber types.Skeletal muscle tissue is that pig body is main Part, more than the 40% of hog on hook weight can be accounted for, it grows to have with growth speed of pigs and closely contacted.Muscle fibre As the main component of musculature, the difference of its type is to influence one of key factor of pig muscle quality, slow type fiber ratio The high pork of example has relatively good m eatquality.Therefore, Six1 is an influence pig growth and the important candidate of Meat Quality Effective molecular labeling of influence pig important economical trait is there may be in gene, its nucleotide sequence.

The content of the invention

It is an object of the invention to develop to can be applied to the molecular labeling of pig production character assist-breeding, simplify the production traits The process of genetic improvement, accelerates production traits genetic improvement process.Identified first from Animal muscles development key gene Six1 Go out potentially influence the hereditary variation site of pig production character, then set up the easy molecule mark in corresponding hereditary variation site Remember detection method, and the application effect of molecular labeling is verified in business swinery, as a result show developed molecule Mark can be applied to the molecular marking supplementary breeding of at least one of Pock Color, weaning weight, carcass weight and chest back fat thickness character.

The purpose of the present invention can be achieved through the following technical solutions：

A kind of molecular labeling related to pig production character, the molecular labeling is selected from least one of (1)~(2)：

(1) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 2, in sequence table SEQ ID NO：Of sequence shown in 2 There is 1 A11-G11 mutation at 11bp, cause HindII-RFLP polymorphisms；

(2) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 13, in sequence table SEQ ID NO：Of sequence shown in 13 There is 1 (AC) n microsatellites variation at 11bp, n represents (AC) repeat number.

The above-mentioned molecular labeling related to pig production character, its pig production character related to the molecular labeling (1) For Pock Color；The pig production character related to the molecular labeling (2) is weaning weight, carcass weight and chest back fat thickness character.

The present invention is translated according to pig Six1 full length genes mRNA sequence (NM_001199718) in ncbi database first Beginning site ATG position, downloaded from GenBank databases from No. 1 chromosomal nucleotide sequence of pig before Six1 Gene As TG about 2kb promoter sequence SEQ ID NO：1.10 pairs of target area amplification sequencing primers are designed according to the nucleotide sequence downloaded (F1/R1-F10/R10) (such as table 1), it is further of the invention to utilize 10 pairs of designed primers by two generation sequencing technologies to 500 Head Pietrain × Duroc × length resource population, and from 7 different pig kinds in vain × great Bai (skin × Du × length × big), including Great Bai, long white, painted face in Beijing opera, Mei Shan, the husky rhizome of Chinese monkshood, rice pig and Suhuai pig, have carried out target area sequencing in 144 individuals altogether. The sequence obtained finally identifies 12 hereditary variation sites altogether by sequence alignment in Six1 gene nucleotide series, It is located at A/G variations at the 1st introne 1 595b respectively to make a variation with A/G at 1648bp, its sequence signature such as SEQ ID NO：2, SEQ ID NO：3；The G/A variations of+294bp places, the C/T variations of -136bp places, the T/C variations of -157bp places, the A/G variations of -379bp places, - A/G makes a variation at 639bp, the C/T variations of -1363bp places, the G/A variations of -1434bp places, the T/C variations of -1449bp places, -1480bp places AGA deletion mutations, its sequence signature is such as with SEQ ID NO：4, SEQ ID NO：5, SEQ ID NO：6, SEQ ID NO：7, SEQ ID NO：8, SEQ ID NO：9, SEQ ID NO：10, SEQ ID NO：11, SEQ ID NO：12；And -1030bp places (AC) n microsatellites make a variation, and its sequence signature is such as with SEQ ID NO：13.A/G variations cause at wherein the 1st introne 1 595b HindII-RFLP polymorphisms, and to SEQ ID NO：A/G variations are established at the introne 1 595b of Six1 genes the 1st in 1 sequence Utilize restriction endonuclease gene classifying method PCR-HindII-RFLP.The hereditary variation position obtained according to sequencing and digestion Point polymorphism genotypic results, and the correlated traits of experiment colony are recorded, and utilize general linear model (General Linear Model, GLM) analysis is associated to hereditary variation loci polymorphism and pig important economical trait, finally it is determined 2 The individual effective molecular labeling that can be applied to pig production character seed selection.The two kinds of molecular labelings invented according to the present invention can be used for In pig production character marker assisted selection.

Sequence table SEQ ID NO：1 is that the pig Six1 gene groups that the present invention is downloaded from ncbi database refer to sequence Column information.

Sequence table SEQ ID NO：2 be that A/G variations are upper at pig Six1 genes the 1st introne 1 595b of the invention identified Point downstream 10bp sequence signatures (GAACAGGACA [A/G] TTGACTTGAT).

Sequence table SEQ ID NO：3 be A/G variant sites at pig Six1 genes the 1st introne 1 648bp of the invention identified Upstream and downstream 10bp sequence signatures (TCGTCCCCTG [A/G] TCATTTTCTT).

Sequence table SEQ ID NO：4 be the pig Six1 genes+294bp places G/A variant sites upstream and downstream that the present invention is identified 10bp sequence signatures (CAGGCCCCA [G/A] CCATGTCGAT).

Sequence table SEQ ID NO：5 be C/T variant sites upstream and downstream at the pig Six1 gene-1s 36bp of the invention identified 10bp sequence signatures (GCTCCACAGC [C/T] TCGCCCTCCC).

Sequence table SEQ ID NO：6 be T/C variant sites upstream and downstream at the pig Six1 gene-1s 57bp of the invention identified 10bp sequence signatures (CACCACTCCC [T/C] ACCCGCCCCC).

Sequence table SEQ ID NO：7 be the pig Six1 genes -379bp places A/G variant sites upstream and downstream that the present invention is identified 10bp sequence signatures (GACGGGGACA [A/G] GGGTACAGAG).

Sequence table SEQ ID NO：8 be the pig Six1 genes -639bp places A/G variant sites upstream and downstream that the present invention is identified 10bp sequence signatures (TGAGAACATG [A/G] GAAGTACTCT).

Sequence table SEQ ID NO：9 be C/T variant sites upstream and downstream at the pig Six1 gene-1s 363bp of the invention identified 10bp sequence signatures (TAATCAGAG [C/T] TAACTAGAGC).

Sequence table SEQ ID NO：10 be G/A variant sites upstream and downstream at the pig Six1 gene-1s 434bp of the invention identified 10bp sequence signatures (CAGTCACTAA [G/A] AGGACACATT).

Sequence table SEQ ID NO：11 be T/C variant sites upstream and downstream at the pig Six1 gene-1s 449bp of the invention identified 10bp sequence signatures (AGAGTTTGTT [T/C] CTCACAGTCA).

Sequence table SEQ ID NO：12 be the AGA deletion mutations site at the pig Six1 gene-1s 480bp that the present invention is identified Upstream and downstream 10bp sequence signatures (GCAGATTTAA [AGA/---] CAAGCAAGTT).

Sequence table SEQ ID NO：13 be (AC) the n microsatellites variation at the pig Six1 gene-1s 030bp that the present invention is identified Site upstream and downstream 10bp sequence signatures (ATTGCAATCT [ACACACACACACACACACACACACACACACACACACAC] TTCTTTATTT)。

The target area sequencing primer of table 1 and PCR-RFLP genotyping primers

At least one of the primer pair of the molecular labeling base mutation above-mentioned for detecting, including following (1)~(3)：

(1) detection sequence table SEQ ID NO：A/G variations (i.e. such as SEQ ID NO at 11bp in 2：Six1 bases shown in 1 Because the 1st introne 1 595b locate A/G make a variation) forward and reverse amplification sequencing primer DNA sequence dna it is as follows：

SEQ ID NO：2

Forward primer (F10)：CTCTGGACACCACTTCCCATGAC (is shown in Table 1)

Reverse primer (R10)：ACAAGCACGCATTTAGAAGTCACT (is shown in Table 1)

(2) detection sequence table SEQ ID NO：In 2 sequences the PCR-HindII-RFLP Genotypings of base mutation just, The DNA sequencing sequence of reverse primer is as follows：

SEQ ID NO：2

Forward primer (F11)：CCGTCCGTCCTTTAAGTCAG (is shown in Table 1)

Reverse primer (R11)：AGCACGCATTTAGAAGTCAC (is shown in Table 1)

(3) detection sequence table SEQ ID NO：The variation of (AC) n microsatellites is (i.e. positioned at such as SEQ ID NO in 13 sequences：1 institute The Six1 gene promoter areas (areas of -1029bp--1100) shown (AC) n microsatellites variation) forward and reverse amplification sequencing primer DNA sequencing sequence it is as follows：

SEQ ID NO：13

Forward primer (F3)：GGACATCTCTCATTCTCTGGCAGT (is shown in Table 1)

Reverse primer (R3)：CCCTGGGAACTTCACACGAAAGG (is shown in Table 1)

The SEQ ID NO invented according to the present invention, the present invention：2 and SEQ ID NO：2 kinds of molecular labelings in 13 sequences It can be used in pig marker assisted selection.

A kind of preparation method of the molecular labeling related to pig production character, is at least one of following (I)~(III)：

(I) SEQ ID NO are included with R10PCR amplifications using described primers F 10：There is 1 at the 11bp of 2 sequences The genetic fragment of A11-G11 mutation, then carries out Genotyping using sequencing technologies；

(II) SEQ ID NO are included with R11PCR amplifications using described primers F 11：There is 1 at the 11bp of 2 sequences The genetic fragment of A11-G11 mutation, then carries out digestion Genotyping using HindII restriction enzymes under the conditions of 37 DEG C；

(III) SEQ ID NO are included with R3PCR amplifications using described primers F 3：13 sequences (AC) n microsatellites make a variation, so Afterwards Genotyping is carried out using sequencing technologies.

Application of above-mentioned molecular labeling, primer pair and the preparation method in pig production character assist-breeding.Described pig The production traits is only few a kind of character in Pock Color, weaning weight, carcass weight and chest back fat thickness.

The beneficial effects of the invention are as follows：

For the economic characters of high heritability, traditional traditional breeding method system can obtain preferable Breeding Effect, But for low-heritability traits, especially for the Meat Quality for needing to determine after butchering, such as color traits utilize conventional breeding When technical system carries out seed selection, it is high to there is cost, genetic progress slowly shortcoming.The present invention goes out from pig Six1 identified for genes In two effective molecular labeling sites, the A/G variant sites that one is located at pig Six1 gene First Introns 1595bp, Another molecular labeling is located at (AC) n microsatellite variant sites of Six1 gene promoter areas (- 1029bp--1100bp areas) On, establish 3 kinds of convenient and reliable pleiomorphism detecting methods for two variant sites.At present, we become to two heredity The application effect of ectopic sites is verified that the result is shown in the experiment swinery of 500 scales, what the present invention was identified Two hereditary variation sites and diagnostic method are available for the early stage of pig production character, live body, quick marking supplementary breeding.

More detailed technical scheme is shown in《Embodiment》It is described.

Brief description of the drawings

Fig. 1：It is to utilize A/G abnormal dnas at the introne 1 595b of F3 in the present invention and R3 primers amplification pig Six1 genes the 1st Fragment agarose gel electrophoresis detection figure.In figure：M：DNA molecular amount standard (DL500:500,400,300,200,150,100 Hes 50bp)；1 and 2 swimming lanes：329bp amplified productions.

Fig. 2：It is A/G variations tri- kinds of bases of PCR-HindII-RFLP at the introne 1 595b of pig Six1 genes the 1st in the present invention Because of type electrophoresis pattern.In figure：M：DNA molecular amount standard (DL500:500,400,300,200,150,100 and 50bp)；1 and 2 swimming Road：AG genotype；3 and 4 swimming lanes：GG genotype；5 swimming lanes：AA genotype.

Fig. 3：Being should on (AC) n microsatellites variation influence in pig Six1 gene promoters in two kinds of cell line in the present invention The Dual-Luciferase activity analysis result of gene promoter activity.In figure：pGL3-Basic-(AC)₁₉With pGL3-Basic- (AC)₂₂Situation that (AC) n microsatellite length be respectively 19 and 22 repetition is represented in initiating sequence respectively, and * * * represent P< 0.001。

Fig. 4：It is the Six1 gene expression doses to (AC) n different genes individual in pig Six1 gene promoters in the present invention Testing result.19/19,19/22 and 22/22 to represent in initiating sequence (AC) n microsatellite length respectively be respectively 19 repetitions Homozygous genotype is individual, the heterozygous genotypes of 19 and 22 repetitions are individual, and the homozygous genotype of 22 repetitions is individual, * * tables Show P<0.01, ns represents that difference is not notable.

Embodiment

Embodiment 1:(preparating example)

1st, pig Six1 gene genetics variant sites are identified

(1) reference sequences are downloaded and design of primers

Pig Six1 genome sequence SEQ ID NO are downloaded from ncbi database：1(https:// Www.ncbi.nlm.nih.gov/), including extron, introne, and proximal promoter subsequence, according in ncbi database Pig Six1 full length genes mRNA sequence (NM_001199718) determines transcription and translation initiation site, the handing-over of introne extron Place, proximal promoter initiation site etc..According to the nucleotide sequence downloaded, design 10 pairs of amplifications using primer-design software and survey Sequence primer (such as table 1).

(2) sample multi-PRC reaction

It is common with from 7 different pig kinds, including great Bai, long white, painted face in Beijing opera, Mei Shan, the husky rhizome of Chinese monkshood, rice pig and Suhuai pig 144 genes of individuals group DNA of meter are template, and performing PCR amplification is entered with the multi-PRC reaction condition after optimization.

Reaction system：Reverse system is 20uL, including 1 × PCR buffer (Takara), 2mM Mg²⁺, 0.2mM dNTP, 0.1 μM of forward and reverse primer/each pair primer, 1U HotStarTaq polymerase (Takara), 2 μ L DNA profilings；

Response procedures：95℃(2min)；94 DEG C (20s), 63 DEG C -0.5 DEG C/per cycle (40s), 72 DEG C (1min), 11cycles；94 DEG C (20s), 65 DEG C (30s), 72 DEG C (1min), 24cycles；72℃(2min).

(3) sequencing library is built

By same sample, different fragments pcr amplification product carries out mixed in equal amounts, adds special signature's sequence and completes library Build (Illumina, San Diego, CA, USA), mould then is sequenced using 2 × 300bp of Illumina Miseq sequenators Formula realizes two-way sequencing to purpose fragment.

(4) hereditary variation site is identified

The detection in hereditary variation site is carried out using GATK and Varscan softwares to obtained sequencing data, and to being reflected Fixed hereditary variation site is annotated, and finally 12 something lost are identified altogether in Six1 gene nucleotide series by comparing analysis Progress of disease ectopic sites.

2nd, pig Six1 gene genetics variant sites analysis of genetic diversity

According to the hereditary variation site information identified, we exist to the 12 mononucleotide hereditary variation sites identified 7 different pig kinds, including great Bai, long white, painted face in Beijing opera, Mei Shan, the husky rhizome of Chinese monkshood, rice pig and Suhuai pig, the altogether something lost in 144 individuals Diversity is passed to be analyzed, as shown in table 2 for 12 mononucleotide hereditary variation sites 7 different pig kinds genotype with Gene frequency analysis result.As a result show, A/G variant sites at pig Six1 gene First Introns 1595bp etc. In Chinese native pig breed with there is obvious difference in position gene frequency, external bacon hogs kind is entirely in external bacon hogs kind C allele, gene frequency is 1, and two kinds of allele of Chinese native pig breed are all present, but T allele is accounted for definitely Advantage, this also imply that the hereditary variation site is probably to influence the important site of China and foreign countries' pig kind Relevant phenotype difference, and has There is the value of potential molecular labeling application.

Analysis of genetic diversity of the table 2Six1 gene mononucleotide hereditary variation sites in different pig kinds

For (AC) n microsatellite variant sites in Six1 gene promoter areas (areas of -1029bp--1100), detected 7 different pig kinds in detect 9 kinds of allele altogether, corresponded to 15 kinds of genotype, wherein external bacon hogs kind with (AC)₁₉With (AC)₂₂Corresponding (AC)_19/19, (AC)_19/22With (AC)_22/22Based on three kinds of gene open forms, and Chinese native pig breed with (AC)₂₀With (AC)₂₂Allele is corresponding (AC)_20/20, (AC)_20/22With (AC)_22/22Based on three kinds of gene open forms (table 3).It is whole For body, there is obvious difference in genotype with gene frequency in Chinese native pig breed and external bacon hogs kind, in The genetic diversity of state's local pig breed is abundanter than external bacon hogs kind, and this also imply that the hereditary variation site is probably shadow Ring the important site of China and foreign countries' pig kind Relevant phenotype difference, and the value applied with potential molecular labeling.

Analysis of genetic diversity of pig Six1 gene promoters (CA) the n microsatellite locus of table 3 in different pig kinds

Embodiment 2 (Application Example 1)

1st, the foundation of molecular labeling diagnostic method

(1)SEQ ID NO：The foundation of 2 sequence variations site PCR-RFLP diagnostic methods

For sequence table SEQ ID NO：A/G variations (i.e. sequence table SEQ ID at the introne 1 595b of Six1 genes the 1st in 1 NO：A/G makes a variation at 11bp in 2), we are established to the simple and effective for the site using restriction enzyme HindII PCR-HindII-RFLP methods of genotyping.SEQ ID NO will be included first：The μ L of PCR primer 5 of 2 variant sites, are utilized Restriction enzyme HindII 0.3-0.5 μ L (3-5U), add 1 μ 10 × Buffer of L, supplement distilled water is to 10 μ L, by sample Centrifuged after mixing, 37 DEG C of incubators place 6-12h and carry out digestions, digestion result is detected with 2.5% agarose gel electrophoresis, with solidifying Glue imaging system is taken pictures, and records genotype.

Obtain including SEQ ID NO with F11 and R11 amplification pig genomic DNAs：The introne of Six1 genes the 1st in 1 sequence The 329bp specific amplifieds fragment (see Fig. 1) of A/G variant sites at 1595b, the PCR primer of amplification produces three through HindII digestions Genotype is planted, when the site is A, it is impossible to by HindII digestions, only one of which fragment after digestion, length is 329bp (A equipotentials Gene)；When the site is G, HindII enzymes can be by PCR primer digestion into two fragments, and length is respectively 210bp and 119bp (G allele).The HindII-RFLP digestion genotyping results of pig Six1 genes are as shown in Figure 2.AA genotype only has one 329bp bands；GG genotype has 210bp and the bands of 119bp bis-, and AG genotype has the band of 329bp, 210bp and 119bp tri-.With 2.5% agarose gel electrophoresis detection can significantly differentiate genotype (see Fig. 2)

(2)SEQ ID NO：The foundation of diagnostic method is sequenced in (AC) n microsatellites two generations of variation in 13 sequences

Obtain including SEQ ID NO with F3 and R3 amplifying genom DNAs：The about 250bp of 13 (AC) n microsatellite variant sites Specific amplified fragment, the present invention has carried out genotype detection using two generation sequence measurements to the site.For SEQ ID NO：13 (AC) n microsatellites variant sites, spy of the Genotyping detection with high flux, high accuracy is carried out using two generation sequence measurements Point.Therefore, applicant devises pair of primers F3 and R3, and with F3 and R3, this is expanded to primer comprising SEQ ID NO：13(AC) The fragment of n microsatellite variant sites.Special signature's sequence construct library is added to the fragment expanded, Illumina is utilized 2 × 300bp sequencings pattern of Miseq sequenators realizes two-way sequencing to purpose fragment.

2nd, trait associations are analyzed

Utilize set up SEQ ID NO：2 sequence variations site PCR-RFLP diagnostic methods and SEQ ID NO：13 sequences In (AC) n microsatellites make a variation two generations sequencing diagnostic method, we to pig Six1 gene First Introns 1595bp at A/G variation (AC) n microsatellites variant sites of site and promoter region (areas of -1029bp--1100) are in skin × Du × length × big business swinery In carried out Genotyping, such as table 4 and table 5 show genotype of two sites in the business swinery of skin × Du × length × greatly And gene frequency analysis result.A/G variant sites at Six1 gene First Introns 1595bp the skin detected × Succeed 823 individuals of parting in Du × length × big business swinery, and testing result shows that AA genotype is 33, AG genotype 263 be head, and GG genotype 527 is head.And (AC) n microsatellites variant sites of promoter region (areas of -1029bp--1100) are in institute Succeed 509 individuals of parting in skin × Du × length of detection × big business swinery, and testing result shows that (AC) n microsatellites become There are 6 kinds of genotype, (AC) in skin × Du × length × big business swinery in ectopic sites₁₉/(AC)₂₄(19/24) genotype 3, (AC)₂₀/(AC)₂₂(20/22) genotype 13, (AC)₁₉/(AC)₂₀(19/20) genotype 34, (AC)₁₉/(AC)₁₉(19/ 19) genotype 50, (AC)₂₂/(AC)₂₂(22/22) genotype 131, (AC)₁₉/(AC)₂₂(19/22) genotype 278.With Upper result shows that two variant sites have obvious polymorphism in business swinery, the potential valency with molecular markers development Value.

Polymorphism of the A/G variant sites in skin DLY at table 4Six1 gene introns 1595

Polymorphisms of the table 5Six1 gene promoters STR in skin DLY commodity swinery

On this basis, we in skin × Du × length × big business swinery to two hereditary variation sites and pig production character Association analysis is carried out, the production traits for association analysis includes birth weight, carcass weight, leaf fat weight, the shoulder thickness of backfat, chest waist The thickness of backfat, waist recommend the thickness of backfat, the triadic mean thickness of backfat, intramuscular fat content, yellowish pink value a*, yellowish pink value L*, yellowish pink value b*, dehydration Rate.Applicant carries out single mark variance using SAS statistical softwares (SAS Institute Inc, Version 8.0) GLM programs Analysis, according to the skin × Du × length gathered × big F₂For resource population information, the mark that such as places an order is set up for the different production traits Remember correlation model：

Model 1：Y_ijkl=μ+Genotype_i+Sex_j+Sire_k+Dam_l+e_ijkl

Model 2：Y_ijkl=μ+Genotype_i+Sex_j+Sire_k+Dam_l+d_ijklA_ijkl+e_ijkl

Model 3：Y_ijklm=μ+Genotype_i+Sex_j+Sire_k+Dam_l+Batch_m+b_ijklmX_ijklm+e_ijklm

Model 4：Y_ijklm=μ+Genotype_i+Sex_j+Sire_k+Dam_l+Batch_m+c_ijklmZ_ijklm+e_ijklm

Model 1 is suitable for birth weight；Model 2 is suitable for weaning weight；Model 3 is suitable for Meat Quality；Model 4 is suitable for trunk Body weight；Y_ijklOr Y_ijklmFor trait phenotypes value；μ is colony's average；Genotype_iFor genotype effects, Sex_j、Sire_k、Dam_l、 Batch_mFor fixed effect, respectively sex, butcher batch, male animal and dam effect；d_ijklWeaning weight is returned for Weaning Age Return coefficient, A_ijklFor Weaning Age；b_ijklmFor regression coefficient of the carcass weight to Meat Quality, X_ijklmFor carcass weight；c_ijklmTo slaughter Age in days is killed to the regression coefficient of carcass trait, Z_ijklmFor slaughter age；e_ijklOr e_ijklmFor residual error effect.

In addition, for single base variant sites, calculating additive effect of gene and dominant effect using reg programs, and carry out Significance test, additive effect represents AA, AG and GG genotype with -1,0,1 respectively, and dominant effect is represented with 1, -1,1 respectively AA, AG and GG genotype.

As shown in table 6, analysis result shows association analysis result, and the A/G variant sites at First Intron 1595bp are more State property is in significantly correlated (P with yellowish pink red scale value a* values<0.05), imply the genetic improvement that the site can be used for color traits. In addition, analysis result is shown, (AC) n microsatellite variant sites polymorphisms are in significantly correlated (P with weaning weight and carcass weight< 0.05) it is in, extremely significantly correlated (P with chest back fat thickness<0.01) (such as table 7)；In addition, (AC) n microsatellite variant sites Polymorphism, in potential significantly correlated, imply the microsatellite locus and can use with average backfat thickness, intramuscular fat, and percentage of water loss In the genetic improvement of pig associated production character.

A/G variant sites polymorphism and trait associations analysis result at the pig Six1 gene First Introns 1595bp of table 6

Note：Numerically the identical expression difference of target letter is not notable, and when letter is different, capitalization represents that difference is extremely notable (P<0.01), lowercase letter indication difference significantly (P<0.05)；Additive effect negative value represents that allele reduces trait phenotypes value, Its subscript * represents P<0.05, * * represents P<0.01.

The association analysis result of pig Six1 gene promoters (CA) the n microsatellite polymorphisms of table 7 and the production traits

Note：Numerically the identical expression difference of target letter is not notable, and when letter is different, capitalization represents that difference is extremely notable (P<0.01), lowercase letter indication difference significantly (P<0.05).

Embodiment 3：(application example 2)

1、SEQ ID NO：Six1 gene promoters (AC) n microsatellite variant sites Assay of promoter activity is carried in 13 sequences Body is built

Hereditary variation site primer result shows, (AC) n in pig Six1 gene promoter areas (areas of -1029bp--1100) Microsatellite variant sites co-exist in the allele of 4 types in the skin × Du × length detected × big business swinery, altogether group Into 6 kinds of genotype (such as table 5), wherein with (AC)₁₉/(AC)₁₉(19/19), (AC)₂₂/(AC)₂₂(22/22) with (AC)₁₉/(AC)₂₂ (19/22) based on genotype.In order to further prove the regulation and control machine of Six1 gene promoter areas (AC) n variation influence production traits Reason, we demonstrate the influence that (AC) n microsatellites make a variation to promoter activity using Dual-luciferase reportor systerm.

(1) according to genotypic results, we are with (AC)₁₉/(AC)₁₉(19/19) with (AC)₂₂/(AC)₂₂(22/22) gene Type genes of individuals group DNA is template, and performing PCR amplification is entered in design upstream with anti-sense primer, and (upstream is respectively with anti-sense primer GGTACCATTGTTCGGTGGTGCTGAG with CTCGAGAAAGGCCGCTACTATTTGG, at every sense primer 5' end all Introduce KpnI restriction enzyme sites (following dashed part is represented) and corresponding protection base (being represented with shadow region)；In anti-sense primer 5' ends addition XhoI restriction enzyme sites (following dashed part is represented) and corresponding protection base (being represented with shadow region).)；By PCR The purified rear clone that reclaims of amplified fragments determines that sequence is complete to cloning vector pMD19-T (Takara companies) through cloning and sequencing Total correctness, different genotype cloning vector is respectively designated as pMD19-T- (AC)₁₉With pMD19-T- (AC)₂₂。

(2) Assay of promoter activity destination carrier is built

First with restriction enzyme KpnI and XhoI (Thermo Scientific companies) to cloning vector pMD19- T-(AC)₁₉With pMD19-T- (AC)₂₂Double digestion is carried out, and the pcr amplification product fragment after digestion is subjected to purifying recovery；Together Sample reports that empty carrier pGL3-Basic (Promega companies) is carried out using restriction enzyme KpnI, XhoI to Dual-Luciferase Double digestion, and the pGL3-Basic empty carriers after double enzymes are subjected to purifying recovery；Secondly, the PCR amplifications after double digestion is reclaimed Product segments-segment is connected on the pGL3-Basic carriers after double digestion, determines that sequence is completely correct through cloning and sequencing, constructed Carrier be respectively designated as pGL3-Basic- (AC)₁₉With pGL3-Basic- (AC)₂₂。

2、SEQ ID NO：The variation of Six1 gene promoters (AC) n microsatellites influences on promoter activity in 13 sequences

After sequence confirms correctly through sequencing, culture is enlarged to positive colony, and extract plasmid.Plasmid extraction is used The plasmid extraction kit of OMEGA companies is extracted, and plasmid purity utilizes NanoDrop with concentration purity 2000Spectrophotometer (NanoDrop companies of the U.S.) detects that the OD260/OD280 values for the sample surveyed are in 1.8- 2.0 just can be used for follow-up experiment.

(1) cell culture and inoculation

Cell culture carries out conventional cell culture using 10% hyclone (Gibco) nutrient solution, will be thin before transfection Cell density is diluted to 1-4 × 10 with growth medium after born of the same parents' digestion⁵Individual/ml, and take 500 μ l cell suspension inoculations to 24 holes In culture plate, each sample carries out at least three and repeats experiment.Applicant is in two kinds of cell lines, including porcine kidney cell line (PK15) With being analyzed in mouse muscle-forming cell (C2C12) starting activity.

(2) cell transfecting

A. the preparation of transfected plasmids, by purpose plasmid pGL3-Basic- (AC)₁₉With pGL3-Basic- (AC)₂₂Plasmid is equal 0.2 μ g/ μ l are diluted to, internal reference plasmid is diluted to 0.02 μ g/ μ l, then take the plasmid and 0.05 μ g internal references plasmid of 1 μ g mesh to mix Close (20：1 ratio), gently blow and beat uniform.Take respectively in 1 μ g pGL3-Basic and pGL3-Control and 0.05 μ g simultaneously Join plasmid mixing, be respectively used to negative and positive control.

B. the plasmid after being mixed in (a) is diluted with 50 μ l Opti-MEM serum free mediums (Gibco), gently mixed, Room temperature places 5min.

C. appropriate Lipofectamine is taken^TM3000 lipofectamines (Invitrogen companies), with 50 μ l's Opti-MEM serum free mediums dilute, and gently mix, and room temperature places 5min.Depending on amount of the transfection reagent amount by taken plasmid, Transfection reagent in the present invention：The ratio of plasmid is 2.5：1.

D. mixing plasmid will be diluted in (b) to mix with the lipofectamine diluted in (c), and is gently mixed, Room temperature places 20min.

E. when transfection composite is stood, the cell culture medium that the night before last is inoculated into 24 orifice plates removes, and changes Upper fresh Opti-MEM serum free mediums.

F. the transfection composite prepared in 100 μ l (d) is taken gently to add in 24 orifice plates, gently rocking culture plate answers transfection Compound is evenly distributed in cell culture fluid.

G. Tissue Culture Plate is placed in 5%CO₂Cultivate, changed after 4-6h containing the fresh of serum in 37 DEG C of cultures in incubator Culture medium continue to cultivate, the collection of row cell when after 24h.

(3) promoter activity is determined and statistical analysis

Cell is cracked and sample pre-treatments use Dual-LuciferaseReporter Assay System (Promega Company) carry out, concrete operations reference reagent box specification.The determination of activity of sample Dual-Luciferase is in Glomax 20/ Carried out on 20luminometer platforms (Promega companies), Relative Promoter activity is expressed as：Fluorescent luciferase activity (Firely luciferase activity)/renilla luciferase activity (Renilla luciferase activity).It is aobvious Work property is examined to enter using the non-matching sample T methods of inspection (Unpaired-Samples T test) carried in the softwares of SPSS 20.0 OK, P<0.05 thinks that promoter activity difference reaches the level of signifiance, P<0.01, which is considered promoter activity difference to reach extremely, shows Write.Promoter function analysis result shows, pGL3-Basic- (AC)₁₉Startability pole is substantially less than pGL3-Basic- (AC)₂₂ (Fig. 3), same result is obtained in the two kinds of cell models used, is shown that Six1 gene promoters (AC) n is micro- and is defended Star variation can significantly affect the transcriptional activity of the gene.

Embodiment 4：(application example 3)

1st, (AC) n different genotypes genes of individuals expression variance analysis

It can be significantly affected on the basis of Six1 gene promoter activities specify that (AC) n makes a variation, in order to further true Fixed (AC) n variations and the relation of Six1 gene transcript expressions, we make a variation from skin × Du × length × big business swinery according to (AC) n Genotyping result has been picked out (AC) at random₁₉/(AC)₁₉(19/19), (AC)₂₂/(AC)₂₂(22/22) with (AC)₁₉/ (AC)₂₂(19/22) three kind of genotype individuals is each about 25.Extraction (the Invitrogen public affairs of total serum IgE are carried out using TRIzol reagents Take charge of product), utilize Prime Script^TMReagent carries out the synthesis (TaKaRa Products) of the chains of cDNA first, then utilizes Real-time PCR carry out gene expression dose detection.So PCR reactions perform 3 repetitions, gene relative expression levels use △ △ Ct methods are calculated, and gene relative expression levels' correction is carried out using hprt gene as reference gene.Utilize GraphPad Prism carry out graph making, utilize the one-way analysis of variance (One-way ANOVA) in the softwares of SPSS 20.0 Statistical analysis is carried out, and Multiple range test, P are carried out using most rigorous Bonferroni methods<0.01 thinks gene differential expression The pole level of signifiance is reached.Gene expression dose testing result shows that the expression of 22/22 genotype individuals will be significantly higher than 19/22 genetic entities (P<0.00003), and pole is significantly higher than 19/19 genotype individuals (P=0.006), and 19/19 and 19/22 Significant difference (Fig. 4) is not present in genotype individuals expression.Result of study above again demonstrates Six1 gene promoters In (AC) n microsatellites variation can significantly affect the expression of Six1 genes, its genetic polymorphism can as influence pig associated production The molecular labeling of shape is applied to the molecular marking supplementary breeding of pig.

SEQUENCE LISTING

<110>Agricultural University Of Nanjing

<120>The preparation and application of the Six1 gene molecule marker related to pig production character

<130> 2017

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 5845

<212> DNA

<213>Pig

<221>Six1 genes

<400> 1

attttaaaac catataaata atataaggag aaacccacac attgcttctc ctcccactcc 60

cacttatgtc tcaatatgta aattgttcgg tggtgctgag ctcttgcaga tttaaagaca 120

agcaagttta aagaatctag agtttgtttc tcacagtcac taagaggaca catttaagtc 180

aaaagcattt ttgaaatgtg cttaaaattc tgttttgaac ttatctaatc agagctaact 240

agagccataa agtcactatc tttctatcct ttggctcccc ttatctcctt gcattgttct 300

tatctcttgc cagtctcagc tactgacaat agattatctt tgtttagaga tgctaattgt 360

tttcaaaatc tttttaataa ttgctttgga acaaggtgtg caaatgaggc ttaaaataat 420

tacagtttta ctaagcattc tctacacaaa cacaaaataa cggatgatgg gactgaaatt 480

gtaggacatc tctcattctc tggcagtaaa ataggccagc attgcaatct acacacacac 540

acacacacac acacacacac acacacactt ctttatttaa tcttcttgga gccactgtcc 600

tcctcccttt gagggcagtg tccctctcag aacagggcaa ggattatatt acaaccggag 660

ggctattgga gccggacaca gttttattta atttacactc catccagcgc ctttcgtgtg 720

aagttcccag ggcatttttg ctaatagcaa gtaaagcttc tcggcattcc tcagtagcag 780

aggtggcctc ggaaaatgca actgctcaga tgtggaaact gagcccagag gcctgccgac 840

ttaaagcata gaatattaag ataccgaatc acaaatgaac cctcagcaat tgctcagcca 900

ctatgctcta aacagctctt tgcgctgtcc caaatagtag cggccttttg agaacatgag 960

aagtactctg cagttgggag tattcaagtc ctttggaggg cggtgggggc aggagggaag 1020

agaggagaaa ttaggtttgg ctcctagagc tgaagccatt cttccagcgg ctgcagtccg 1080

gagcactgat ctgcttgctc ttgtattatt tatcgcgtac tctaagctat tcgtcaatcc 1140

attacttatt cattccaaca ttaaggtaat taaatatgag ctcaccggga aagggcgagt 1200

ctgcaaggga cggggacaag ggtacagagg aagagaaggg gaatatgaag gtctggagtc 1260

attgatttgt gcagagatga gagcggctgc ccttagatga cactgcagag ggggctcacg 1320

ttgcagggtc ctgacgtact cacccactcc caccgccccc ggtccccgcg aagcgcctct 1380

ccctcttccc tccccatctc gcttcccacc tctcgcggtc ccactcctcc caccactccc 1440

tacccgcccc cgctccacag cctcgccctc cccgcgcggg gcggcggggc ctcgcggccc 1500

ggccaggcgg cgagcaggct ggccagcccc ggataggcgc cgcccgcagc caatcagcgc 1560

gccggggaca ctgcgcgctt taaggcagct gcgggtagaa cagaaaccaa gttccccggc 1620

aacgagcagc atccacccgg cgggagaccg aagcctgaga ggcccgaaaa gctgcggagt 1680

gagccggccg ctccctgtcc agtgcgcgcc ctactcattt tcttctttgg ccggtcgtcc 1740

ccaagtcttg acttgtcttt ccgcctgcat aaagcctcgg gagggggtta ggagcagccg 1800

cgccccccct tcccagcggc tgccgccccc ggctttttcg gctctgctcc ctgccgcgtg 1860

cgctccggca gtgcgcttcg gcaggcccca gccatgtcga tgctgccatc gttcggcttc 1920

acacaggagc aagtggcttg cgtgtgcgag gttctgcaac aaggtgggaa cctggagcgc 1980

ctgggcaggt tcctgtggtc gctgccagcc tgcgaccacc tgcacaagaa cgaaagtgtg 2040

ctcaaggcca aggcggtggt cgccttccac cgcggcaact tccgcgagct ttacaagatc 2100

ctggagagcc accagttctc gcctcacaac caccccaagc tgcaacaact gtggctgaag 2160

gcgcactacg tggaggctga gaagttgcgc ggtcggcccc tgggcgcggt gggcaaatat 2220

cgggtgcgcc gaaaattccc gttgccgcgc actatctggg acggcgaaga gaccagctac 2280

tgcttcaagg agaagtcgcg gggcgtgttg cgggagtggt acgcccataa cccctacccc 2340

tcgccgcgtg agaagcggga gctggccgag gccaccggcc tcaccaccac ccaggtcagc 2400

aactggttca agaaccgaag gcaacgagac cgggccgccg aggccaagga aaggtacggg 2460

gtgtgattcc tgacaatcag gggacccggg gaagtcttcc ttcctctccc tctccgcctc 2520

ttcccgccca cccccctccc cgccccaggc accagccacc gcaacggtcc ctgctgcctg 2580

agccagtccg cggggcggcg cccgggcctc cgcggccctg cggcgaaagt tcatgaactc 2640

tgagatcgct tgctcgggat ggggggctcc tctgatttga gcatccgcgc gtgggtttcc 2700

tgggatgtga gtgtcgggaa atgttggcgc taggtttgtt tttcatgcct cccaggctgg 2760

cgggggccag gagtgacggg tggagaatgg gtttctagat aaattagaaa ggaaggtgtg 2820

atcttgagga aagggattca gtagggaggg aggatctcga gcctttttcc tcttgctccc 2880

cagaggagtg gagctggctg aaacttttgt tcggtctccc ctcccgcctc gtcctgcact 2940

cgctctctcc ttgtagtctt ttgggccaga ggtcgcttca tcccgcacta attgtacaaa 3000

agtaggaatc tttcaaaaga aatcaagagt ttggggcgag ttaccgttgc atgtactccc 3060

aggcgcctca agcccagaat ggatcgctcc tcgctggctt cctcgccggt ggccaggcgg 3120

cgggcgcagc aagtctgtcc gctgtcctcg ccgctgccgc cccaggcgag gcagggatgc 3180

ccagtttcat acttctctcg ccctggagct cagagggcaa tcttttcttt atctgtctct 3240

gcctctgccc tcctcgcgcc agcatcccgg ccacctggcc gggctccagc cgggtgttgt 3300

cgcttccgac cgaggctcga ggttcggagg agcgtggggg aggcggcggt ggggagagca 3360

ctccggggag ccgagcaggt tgcgcgaaac ctgagcgccg gggtcggccg ggtttctttt 3420

gttcgcctca gagcctgggg gccgaaggtg tcgccgccct gctcccctct gggcggaaaa 3480

gccaagtctg gataaattca actgaaatag gctttctggg agagagagag attgcgaccg 3540

gtggggaggg agtgggagag accgcaggcc cgggagtggg gaggaggccg agcgctctta 3600

aggtaaaagc ggcgagagcc cgagaaccct ggtttaccag cacccaacgc ccggactgac 3660

gcccggtgcg gcgcgccggc tcccgcagtt gcggggtggt ggagttcccc cgcccggaaa 3720

gggtgcgctg ctgtggatgt cctcagggag tttgggagag ggccgcgagg cacggaggac 3780

tgagccagta acacgggaac agggacagta caattccagt tagacgttag ttgcaaaacg 3840

gccgtccgtc ctttaagtca ggcccgaggg cccactccct gatgaaatcg gagagactgg 3900

ctgcgctccc tgtgtgttta ctcaggtcac tgggccgctc tggacaccac ttcccatgac 3960

ctgccctgtg tctctgaagc cacaacctcc gggcaggggc gggggaaaac ttgtcagtta 4020

attgtttcct cagatgggaa caggacaatt gacttgattc atgcccaaac atctgtgcac 4080

tgggcgctct tcgtcccctg atcattttct tcatcaccga caaccatttg ttggaagttg 4140

ttcacattta gtgacttcta aatgcgtgct tgttgatagg tatttaaaaa taactgcttg 4200

aacaaaaatt ttaaagtaat ttttgtgttg cacataagct ctgctgatct ggctttctaa 4260

aatctggaag aggtaaggac ccagctcccc tttagctccc cttctcaagt ctctgctgcc 4320

tcctcatctc cccagggccc ttggggatga gtgagagggc agagatcctc catttgggtc 4380

aatgactgat tgctcaatgg ccctttccta ctcctagttc tcctccctcc atctcacact 4440

ttttttaact tttttttttt tttttttttt aaccatatgg tgttgtcctc tatttcttag 4500

ggagaacacc gaaaacaata actcctcctc caacaagcag aatcaactct ctcctctgga 4560

agggggaaag ccgctcatgt ccagctcaga agaagaattc tcacctcccc aaagtccaga 4620

ccagaactcg gtccttctgc tgcagggcaa tatgggccac gccaggagct caaactattc 4680

cctcccgggt ttaacggcct cgcagcccac ccacggcctg caagcccacc agcatcagct 4740

ccaggactcc ctgctgggcc ccctcacctc cagtctggtg gacttagggt cctaagaggg 4800

gagggaatgg ggcctcaagc agcgactagg gacacttgta aatagaaatc aggaacattt 4860

ttttgcagct tgtttctggg gttctttgcg cataaaggaa tggtgaactt tcataaatat 4920

cttttaaaaa ctcaaaacca accacagtct caagcttagt gccctcttct ctccaactct 4980

ttccactttt gcatctcccc tcccagatca gggaaaagga agaagaagaa gaagacgaag 5040

aagaagaaac ccaaacaaac aaaaacatcc agtcacccag tcttggttct gggcaatcca 5100

cattgctgct tcagtagtct ctctttctct ctgtccctct cttttttgtt caatgaacag 5160

ggcttacaaa tcaactggat tttaattatt tccttttaat ttatttatgg taaaatgttt 5220

tgagaagagg aaaaggaaat gaaaatgaga gagaagagag agagaaagtc aaaatagtga 5280

aaagagcctc tatcctggga ggaacttgtg ttcttaaggt gaataggaaa aatatgatct 5340

tacaattttt tttgatgggg aaaattaagt ttacattttt catatttagt gttaaacaat 5400

tttatgtaga ttaaaatgaa agaacagtat tgggggaaaa aacaagtgcc taaatgtctt 5460

aatgctctct acataaggca gttagaaata gaagtgacca ttagtaatac aactattttt 5520

gtcaaattta gttggggggt ggttgttgtt tgtttccttg ggttttttct atggcaaact 5580

tttaaaatgt accaatgtga aaaaacactt tcctgaatgc cattacttct catgccctca 5640

aagctttcat atttgtagcc tactcctgtt aaaggtttct cctgttccta gtttctagct 5700

tgcaaaaata cgtaacaaat ctggcaacct ggtatttgtt actaaacagc atgtgttttc 5760

aggtttcttt tctattgtac ctaaaccagt ctaaattaaa acttagtaga accccaaaaa 5820

aaaaaaaaaa aaaaaaaaaa aaaaa 5845

<210> 2

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 2

gaacaggaca [a/g]ttgacttga t 21

<210> 3

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 3

tcgtcccctg [a/g]tcattttct t 21

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence

<221>

<400> 4

caggcccca[g/a] ccatgtcgat 20

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 5

gctccacagc [c/t]tcgccctcc c 21

<210> 6

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 6

caccactccc [t/c]acccgcccc c 21

<210> 7

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 7

gacggggaca [a/g]gggtacaga g 21

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 8

tgagaacatg [a/g]gaagtactc t 21

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<221>

<400> 9

taatcagag[c/t] taactagagc 20

<210> 10

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 10

cagtcactaa [g/a]aggacacat t 21

<210> 11

<211> 21

<212> DNA

<213>Artificial sequence

<221>

<400> 11

agagtttgtt [t/c]ctcacagtc a 21

<210> 12

<211> 23

<212> DNA

<213>Artificial sequence

<221>

<400> 12

gcagatttaa [aga/---]caagcaa gtt 23

<210> 13

<211> 58

<212> DNA

<213>Artificial sequence

<221>

<400> 13

attgcaatct acacacacac acacacacac acacacacac acacacactt ctttattt 58

Claims

1. a kind of molecular labeling related to pig production character, it is characterised in that：The molecular labeling is selected from (1)~(2) extremely Few one kind：

(1) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 2, in sequence table SEQ ID NO：At the 11bp of sequence shown in 2 There is 1 A11-G11 mutation, cause HindII-RFLP polymorphisms；

(2) its DNA sequence dna such as sequence table SEQ ID NO：Shown in 13, in sequence table SEQ ID NO：The 11bp of sequence shown in 13 There is 1 (AC) n microsatellites variation at place, and n represents (AC) repeat number.

2. the molecular labeling related to pig production character according to claim 1, it is characterised in that：With the molecular labeling (1) related pig production character is Pock Color；The pig production character related to the molecular labeling (2) is weaning weight, carcass weight With chest back fat thickness character.

3. the primer pair of the molecular labeling base mutation described in test right requirement 1 or 2, it is characterised in that：Including (1)~(3) At least one of：

(1) detection sequence table SEQ ID NO：In 2 at 11bp A/G make a variation forward and reverse amplification sequencing primer DNA sequence dna such as Shown in lower：

Forward primer (F10)：CTCTGGACACCACTTCCCATGAC

Reverse primer (R10)：ACAAGCACGCATTTAGAAGTCACT

(2) detection sequence table SEQ ID NO：The PCR-HindII-RFLP Genotypings of base mutation is forward and reverse in 2 sequences The DNA sequencing sequence of primer is as follows：

Forward primer (F11)：CCGTCCGTCCTTTAAGTCAG

Reverse primer (R11)：AGCACGCATTTAGAAGTCAC

(3) detection sequence table SEQ ID NO：The DNA for the forward and reverse amplification sequencing primer that (AC) n microsatellites make a variation in 13 sequences Sequencing sequence is as follows：

Forward primer (F3)：GGACATCTCTCATTCTCTGGCAGT

Reverse primer (R3)：CCCTGGGAACTTCACACGAAAGG.

4. a kind of preparation method of the molecular labeling related to pig production character, it is characterised in that：Including in (I)~(III) extremely Few one kind：

(I) SEQ ID NO are included with R10PCR amplifications using the primers F 10 described in claim 3：At the 11bp of 2 sequences There is the genetic fragment of 1 A11-G11 mutation, then carry out Genotyping using sequencing technologies；

(II) SEQ ID NO are included with R11PCR amplifications using the primers F 11 described in claim 3：At the 11bp of 2 sequences There is the genetic fragment of 1 A11-G11 mutation, digestion gene is then carried out under the conditions of 37 DEG C using HindII restriction enzymes Parting；

(III) SEQ ID NO are included with R3PCR amplifications using the primers F 3 described in claim 3：13 sequences (AC) n microsatellites Variation, then carries out Genotyping using sequencing technologies.

5. application of the molecular labeling in pig production character assist-breeding described in claim 1 or 2.

6. application of the primer pair in pig production character assist-breeding described in claim 3.

7. application of the preparation method in pig production character assist-breeding described in claim 4.

8. according to any described application in claim 5~7, it is characterised in that：For Pock Color, weaning weight, carcass weight with Application at least one of the chest back fat thickness molecular marking supplementary breeding of character.