CN104232630A

CN104232630A - Two major markers for increasing number of pork ribs and application thereof in pig breeding

Info

Publication number: CN104232630A
Application number: CN201410419679.8A
Authority: CN
Inventors: 任军; 黄路生; 幸宇云; 张志燕; 艾华水
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2012-12-04
Filing date: 2012-12-04
Publication date: 2014-12-24

Abstract

The invention discloses a major marker on pig chromosome 7 for increasing the number of pork ribs and application thereof in boar genetic improvement. A technique of direct electrophoresis after real-time quantitative TaqMan PCR (polymerase chain reaction) technique and PCR amplification is utilized to perform genotype determination on the g.103458678_103458679ins291 major mutant site; and marker assisted selection (MAS) is utilized to select the favorable genotype for individual breed reservation, thereby enhancing the rib number of each individual in commercial pig breed and Chinese local pig breed by about 1 on average, and further increasing the body length (meat yield) of the pig.

Description

Two increase the main effect mark of pig rib number and the application in pig breeding thereof

The application is the application number submitted on December 04th, 2012 is the divisional application of the Chinese invention patent application of the same name of 201210509536.7.

Technical field

The present invention relates to Animal Genetics field, especially relating to two increases the main effect mark of pig rib number and the application in kind of swine improvement thereof.

Background technology

Pig belongs to vertebrate, and the vertebra of pig is divided into cervical vertebra, thoracic vertebrae, lumbar vertebrae, recommends vertebra and tail bone, and the cervical vertebra of pig, to recommend vertebra relative with tail bone number fixing, and thoracic vertebrae and lumbar vertebrae number there are differences, and thoracic vertebrae is commonly called as rib again.Pig rib number is important economic characters, and its heritability is up to more than 0.6.The long significant correlation of trunk of rib number and pig, along with the increase of rib number, the trunk of pig is long also in corresponding growth, and often increase by a root bone number and can make the long increase of body about 80mm, therefore the meat yield of pig also obtains corresponding increase.

Applicant utilizes the extensive white duroc × Erhualian F built early stage ₂sources group, determines 1029 F ₂butcher individual vertebra number phenotype, detected the major gene loci (QTL) No. 7 karyomit(e)s affecting pig rib number by genome-wide screening, the interval size of Primary Location is 900kb.

In view of above background, applicant is by white duroc × Erhualian F ₂sources group, Erhualian × Tongcheng pig F ₂sources group associates (GWAS) and analyzes identical with descendant's homology (IBD) analysis with the full-length genome of Su Tai swinery body, QTL interval is narrowed down to 100kb.To this region carry out sequence of resurveying, the search of polymorphic site, discriminating and and the research of pig rib data/coherency, the gene breeding technology to setting up efficiently and accurately carries out the seed selection work of pig rib number.

Summary of the invention

First object of the present invention is to provide the main effect mark that two increase pig rib number, that is: the genovariation sequence be positioned on pig No. 7 karyomit(e)s shown in sequence table SEQ ID NO:1, and sequence labelling position is the coding mutation of the A2401-C2401 of 2401; In sequence table SEQ ID NO:1, sequence labelling position is the insertion mutation of 291 Nucleotide between 3678 and 3679.Detect this two main effect marks by modern molecular biology technique, judge to carry the individuality increasing rib number favorable allels.

Second object of the present invention is the application that the main effect of increase pig rib number is marked in kind of swine improvement, namely the main effect label information differentiated is utilized to carry out boar seed selection, selection causes the more genotype individuals of pig rib number to be reserved seed for planting, and grows and meat yield with the rib number, the trunk that improve boar.

For this reason, the present invention first aspect provides a kind of main effect mark, and the sequence of this main effect mark is as shown in sequence table SEQ ID NO:1, and the position wherein between this sequence the 3678th and 3679 is the insertion mutation of 291 Nucleotide.

The main effect that the present invention second aspect provides described in the present invention first aspect is marked at too pig of reviving, the west three way cross market pig obtained is hybridized, the application in the pig rib number qualification of the hybridized pig of the hybridized pig of duroc and Erhualian and Erhualian and Tongcheng pig by duroc, landrace and Large White.

First object of the present invention is achieved in that

1, laboratory animal and phenotype test

Swinery body used in the present invention has 4: white duroc × Erhualian F ₂sources group, Erhualian × Tongcheng pig F ₂sources group, revive too pig (50% duroc and 50% Taihu pigs blood relationship) colony and assorted (duroc × landrace × Large White) colony of ternary.

White duroc × Erhualian F ₂sources group with 2 white Duroc boars and 17 Erhualian sows for breed F for generations ₁in generation, select 9 F ₁boar and 59 F ₁sow divides 6 batches to hand over generation 1912 F mutually ₂individuality, wherein 918 F ₂individuality butchers to record rib number phenotypic data at 240 ± 3 ages in days.

Erhualian × Tongcheng pig F ₂sources group obtains 2 F with 1 painted face in Beijing opera boar and 1 Tongcheng sow mating ₁boar and 7 F ₁sow, F ₁offspring hands over rear generation 61 F mutually ₂individuality, all F ₂individuality butchers to record rib number phenotypic data at 60 ages in days.

Too pig of reviving is that only one China cultivates kind, and it is the pig kind (respectively containing the blood relationship of 50% Taihu pigs and 50% duroc) obtained by the cultivation more than 18 generations by Chinese Taihu pigs and west duroc kind; In the present invention, 4 revive too boar and 55 too sow mating of reviving obtain 461 offsprings, butcher to record rib number phenotypic data for wherein 435 at 240 ± 3 ages in days.In addition, the mix sample of market pig and rib number phenotypic data of the ternary of 1403 195 ± 3 ages in days divides 9 batches to obtain from the Jiangxi Guo Hong company limited slaughterhouse being positioned at Nanchang Jiang Xiang.

2, pig full-length genome 60K SNP sentences type

Gather a fritter ear sample from each individuality above-mentioned 4 experimental populations, extract complete genome DNA by standard phenol-chloroform method, unify concentration dilution to 50ng/ μ l after Nanodrop-100 spectrophotometer Detection job, send the happy Mei Tongde company limited in Beijing to carry out pig full-length genome 60K SNP chip (Illumina, the U.S.) genotype according to company standard flow process on Illumina Beadstration platform to judge.In utilizing R language GenABEL to wrap, checkmarker carries out quality control to all sample 60K chip scanning typing data, for SNP recall rate lower than 95%, family Mendelian error rate higher than 0.1, minimum gene frequency be less than 0.1 and Hardy-Weinberg equilibrium significance level higher than 10 ^-6individual information by disallowable.

3, full-length genome association (GWAS) is analyzed

In order to eliminate colony's stratification effect, the present invention adopts the regression analysis of linear mixed model single-point and carries out GWAS analysis in conjunction with the GenABEL software package in R program.For meta-analysis (confluence analysis), the χ of each site in 3 colonies ²value combines and calculates new χ ²value.The marking area of genomic level adopts conservative Bonferrini bearing calibration to determine, namely genome conspicuous level threshold value is 0.05.As shown in Figure 1, as can be known from Fig. 1, the site affecting pig rib number is all positioned on No. 7 karyomit(e)s GWAS analytical results by the analytical results of 3 different groups GWAS and meta-analysis.By the method for LOD (logarithm of likelihood function ratio) value decline 2, according to the fiducial interval that 3 experimental populations are shared, determine in the scope of a 947kb on No. 7 karyomit(e)s by the main effect site affecting rib number that GWAS locates, this region corresponds to the interval of international pig genome reference sequences (10.2 version) No. 7 chromosomal 103.37Mb to 104.31Mb.

4, descendant's homology (IBD) positioning analysis

The auxiliary method for separating and analyzing of mark is adopted to judge white duroc × Erhualian F ₂sources group, Erhualian × Tongcheng pig F ₂the F of sources group, the too pig of reviving ₀and F ₁boar QTL genotype.Every QTL genotype with descendant's phenotype test pig is determined by Z value, and Z value is likelihood ratio ratio L _h1/ L _h0log value, H ₀assuming that diallele QTL genotype is homozygote QQ or qq, H ₁for heterozygote Qq.As Z<-2, QTL genotype is QQ or qq; As Z>2, QTL genotype is Qq; As-2<Z<2, QTL genotype be can not determine.The haplotype of QTL genotype individuals is successfully derived by simwalk2 program construction, and by searching the shared haplotype of Q karyomit(e) to reduce QTL region.For by IBD area reduction in interval little as far as possible, first use 60K high-density SNP chip scan-data to carry out IBD analysis in the present invention, concrete outcome is shown in Fig. 2 A.Be defined between SNP INRA0027623 and ASGA0035500 from this figure, IBD interval.In order to IBD interval is reduced further, after pig genome sequence between SNP INRA0027623 and ASGA0035500 is downloaded by Ensembl website (http://asia.ensembl.org/index.html), every about 20000bp public Primer3.0 or Primer5.0 software design pair of primers (totally 12 pairs of primers, in table 1), pcr amplification is carried out to 35 genotypic individualities of known QTL.In polymerase chain reaction (PCR) reaction system of 25 μ L, comprise 40ng pig genomic dna, 1.0mM MgCl ₂, 0.2mM dNTP, each 10pmol of forward and reverse primer, 2.5 unit archaeal dna polymerases (Taq enzyme) and 1 × PCR buffer (damping fluid) (company is widely collected in Shanghai).PCR adopts Touchdown program, and amplification condition is: 95 DEG C of 5min; 94 DEG C of 30s, 68 DEG C of (each cycle down 0.5 DEG C) 30s, 72 DEG C of 45s, 26 circulations; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s, 14 circulations; Finally extend 10min at 72 DEG C.Pcr amplification product entrusts Shanghai Sheng Gong biotechnology company limited direct Sequencing after adopting QIAquick DNA purification kit (QIAGEN, Hilden, Germany) purifying, and sequencing result utilizes the SeqMan software of public DNAStar to analyze.Again IBD analysis is carried out to the new SNP information obtained, the region that Q karyomit(e) is shared as shown in Figure 2 B, known from Fig. 2 B, QTL interval is accurately positioned in the interval of a 100kb (between SNP103451019T>G and SNP103552163G>A).

In IBD interval, table 1 pig No. 7 karyomit(e) main effect sites, SNP scans primer

5, the sequence of resurveying that object IBD (100kb) is interval

Downloaded the pig genome sequence of about 100kb between SNP103451019T>G and SNP103552163G>A by Ensembl website (http://asia.ensembl.org/index.html), with the online software of Primer3.0 (http://frodo.wi.mit.edu/) design primer (see table 2) to white duroc × Erhualian F2 sources group, revive too in pig and Erhualian × Tongcheng pig F2 sources group genotypic 35 individualities of known QTL to resurvey sequence.In polymerase chain reaction (PCR) reaction system of 50 μ L, comprise 100ng pig genomic dna, 2.5mM MgCl ₂, 0.4mM dNTP, each 20pmol of forward and reverse primer, 2.5 unit archaeal dna polymerases (La Taq enzyme) and 1 × La PCR buffer (damping fluid) (Takara company).Pcr amplification condition is: 94 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 8min, 30 circulations; Finally extend 10min at 72 DEG C.Pcr amplification product entrusts Shanghai Sheng Gong biotechnology company limited direct Sequencing after adopting QIAquick DNA purification kit (QIAGEN, Hilden, Germany) purifying, and sequencing result utilizes the SeqMan software of public DNAStar to analyze.

The primer of the interval sequence of resurveying of table 2 100kb IBD object

6, polymorphic site genotype and the genotypic consistency analysis of QTL

35 that sequence of resurveying are obtained individual polymorphic site genotype, compared with QTL genotype, get rid of its possibility being main effect mutational site by QTL genotype and the genotypic discordance of polymorphic site.35 individualities search 187 polymorphic sites altogether in about 100kb interval, eliminate 183 polymorphic sites altogether by polymorphic site genotype and the genotypic discordance of QTL, finally remain 4 its genotype of polymorphic site and conform to (see table 3) with QTL genotype completely.G.103446231G>A these 4 polymorphic sites are, g.103451235C>T, and g.103458678_103458679ins291 g.103457401A>C, it is respectively the be positioned on the international pig genome reference sequences (10.2 version) in Ensembl website (http://asia.ensembl.org/index.html) No. 7 karyomit(e)s the 103446231st, 103451235, the SNP (mononucleotide polymorphic site) of 103457401 Nucleotide and a clip size between the 103458678th and 103458679 Nucleotide are insertion (ins)/disappearance (deletion) sudden change of 291bp.

Four, table 3 affects mutational site genotype and the genotypic consistency analysis of QTL of pig rib number

WE, white duroc × Erhualian F ₂sources group; ET, Erhualian × Tongcheng pig F ₂sources group; SU, too pig of reviving.

7, the conservative Analysis in 4 candidate's main effect mutational sites

G.103446231G>A, g.103451235C>T, g.103457401A>C and g.103458678_103458679ins291 the present invention carries out conservative Analysis to 4 possible main effect mutational sites.According to having been reported, only there is variation in rib (thoracic vertebrae) quantity in mammiferous pig and sheep, if therefore there is variation in the Mammals of certain mutational site above-mentioned outside pig, sheep, can be excluded as cause and effect sudden change.The present invention compares above-mentioned 4 sequence conservations of candidate's main effect mutational site in pig, ox, dog, horse, people and mouse, the results are shown in Figure 3.G.103446231G>A and g.103451235C>T can learn from this figure, there is variation in two mutational sites in ox, dog, horse, people and mouse, therefore gets rid of these two sites for the sudden change of candidate's cause and effect.

8, the correlation analysis of the sudden change of candidate's cause and effect and rib number phenotype

The present invention is directed to remaining 2 main effect mutational sites and g.103457401A>C and g.103458678_103458679ins291 carry out correlation analysis with pig rib number phenotype.G.103457401A>C main effect mutational site is corresponding to the 103457401st nucleotide site, i.e. the 2401st Nucleotide of following SEQ ID NO:1 sequence on international pig genome reference sequences (10.2 version) No. 7 karyomit(e)s of Ensembl website announcement; G.103458678_103458679ins291 main effect mutational site is corresponding to the 103458678th and 103458679 Nucleotide on international pig genome reference sequences (10.2 version) No. 7 karyomit(e)s of Ensembl website announcement, an insertion (ins) namely between following SEQ ID NO:1 sequence the 3678th and 3679 Nucleotide/disappearance (deletion) sudden change.

SEQ ID NO:1 is the sequence on international pig genome reference sequences (10.2 version) No. 7 karyomit(e)s of announcing of Ensembl website between the 103455001st Nucleotide to the 103459980th Nucleotide.

TTCCTGAACC?AAAATGTACA?AGCAGTTTGA?ATCATATACA?GTGGTCCAGT?GGTCCCCTCT?60

CACCCGTGGC?TCCCCCACCC?CCTTTCCAGA?CCTCTGGCGG?ATGCCTGAAA?CCACAGTTAA?120

TACCAAATCC?TATGTGTAGA?CACCGTTTTC?CCCTATAGAC?AGCATGGAGA?GGCTGAATGG?180

AGGGAGGATT?CCTTTACTTC?CCAGCTGGGA?CAACAGAGCA?GGGCGAGAGA?GCTCATTGTG?240

CTACGGGAAG?AGTGCACAGT?TTAAAAGTCA?TGAGGGTTCA?TTTCTGGGAT?TTGTCATTTA?300

ATGTCTCCCC?ACTGCAGATG?ACTTCAGGGA?ACGGAAAACA?AAGAAAGCCA?CACTGCAGAT?360

ATGGAGGGTC?TACTGTATTA?TTATTTAGAT?ATCATCTATG?TACATAATCC?ATATTATGTA?420

CATACATAGT?AGTCAAAAGT?AGTTTAAAAA?GTTTAACATA?GAATAAAAAT?ATGCACAGAG?480

AGACGAGACA?CAAATACACA?CGGGCAAATG?CACAGAAAAA?GATGAAACTG?CACAGAAAAA?540

AGAACTAGGC?AGATACACAG?AGTGAGGAGA?ACGTGTGAGG?GACTGAAAGG?GAGGACGTGC?600

TGAATTTGCT?CTATTTTTTA?CTCTGTACCG?TTCGTTTGAG?TCTTTTGTAA?GGACAAGGCT?660

TAATTTGTGT?TAAGTCTTAG?AAAAGAAACT?AGATTAGAAA?GAAATGTACC?AAGATATTCA?720

CAATAGCTGT?CTCTAGATAA?TGAGATTAGA?GGTAATTTTT?CCTTCCTATT?ACTTTAAAAA?780

TCCTCCCAAT?TAGTCTTCAT?TTTCCCAATT?AGTAGATAAA?TAAGACTCTT?ACTTTGATAA?840

TGGAAGCACC?AACAAACCAT?TTTTATCACT?ATAGTTTCTT?TGTAAAAAAC?ATACTTTTAA?900

AGATTCTATG?AGCCTTCTAA?AGAACTTTAA?ATTATCATCA?TTGCCTTTTT?AAACTGATAG?960

CAAGACTCCT?TATAAAATGT?CAAAAACAGA?GACATGGAAA?GACGTATCTC?ATAATTTCAT?1020

CTCCTTCTGC?AAAATCACAA?TTCTTTGAAA?CTTTAAAAAT?TCTGCATTTC?TATTAAGAAC?1080

TTTAAAATAA?TTTCAGATTC?ACTGGAGAAG?CTGAGAATGG?GAATATACTT?GCTTTGGGCC?1140

AAGCAGAATC?CTAAATGCTT?TACATTTATC?ATATTGAATC?TACCCAACGA?CTCTCCAGGG?1200

GAAAGAACTC?TCTGGGGTCG?GTGTTATGCC?CCAGGAAGCA?CTAAAGCAGG?GCTCTGAGGT?1260

TTAAACAAGG?TGCCTAAGGC?CATACTCTCT?CAGGGCAGGG?TTGGCATTCA?AATCCAAGTC?1320

CAGGAGGAAA?GCCCAAGCCA?GTGCCTCCCA?CAGCTGCCAT?TGAGGTGGGT?GGGGCTGGTG?1380

TGACTTGGAT?GCAGGACCAG?GATTTGAGTT?CACTTCTGGT?GGCCCCCGAT?CCAGGTCCCT?1440

GAGAATGCGT?GTTTGAGAAA?GGATTTTGAC?TTCGGTGATG?AGTGTAGGTT?CATGACTGGT?1500

GGAGGAGGAA?ACGATAATGC?TAGAGCTGGG?GCATAACCGA?CCTTGTGGTG?AGGGGTGGAG?1560

GTTTCCGTGG?TAGCTGTGGG?TAGGCGCTGA?AGGGGAAAGG?GGGGTCGAAC?TGAAGCCCAG?1620

GAGGGCTGCC?AGCTCCAGCA?CTCCAAAGGG?TTAAGGCCGG?GCAGGAAAGC?CTATGGGGAT?1680

GGGGTAGAGG?GTGGGGGGTG?GGGGGAGCTA?CCACCAAAGG?CTGCGCCATC?TAGCAGCCCT?1740

CACCTCCCCC?ACCAGCAGGG?ACTGGACGGG?CGGAGGGGAC?TAGTTCCTCA?GGGGATTATC?1800

TGAGGCCTCT?GGGCTGAGGT?GAGCTCCGGG?GAGGGTCGGA?AGCCCCAGGT?GCCACGCGCC?1860

CCCACCTTCC?TGTGGGGCTC?AGAAAACTAG?GTGACCTGGG?ATAGGCGCCC?TGACCCCTGA?1920

CAAAAGCATC?TTTGTCCCAG?GCACACTTTG?GGAGTGAGGG?GTGTTGAGCC?CAAGCCTGGG?1980

GGATGCTCAG?ACAAAGGTGG?GGATGGAAGC?CTTTCAGGAA?GGGAATGGGA?CCAGCACCTA?2040

GGCGGGGGCA?GGGAGTTCTC?CAGCCTCCCC?ATCTCGCAGG?GCCTGGGGTG?GGGGAGCGGT?2100

CTCAGGAAGA?GATGCATCCC?TGGGGCTCAG?AGAGCCCATC?ACCCAGGCCA?GTTTCCCCAG?2160

CTCAGCTTCG?CTCTTTCCCC?CTCCCCCCCT?TAGCCTGGCT?TCAGACTCTC?TTTCCTCAGG?2220

TTCTTTCTAT?CTAAACCTTC?TCCTCCCCTC?CCCTCCCCCC?AAACTCTTTG?GAAATCTTTC?2280

AACTTTCTTT?CAAGTTTTCC?TGGGCAGCTC?GGGAATCTGA?GCTAGGTCAG?GGCGCTTTCA?2340

CACCCCCAGC?TGGGTAGAGT?GGACGAGACA?ATAGCTGCTA?TCACTTTACC?ATTGGAAGTG?2400

ACCCCCGAAT?TTGCACTTCT?TTCATCTCGG?GACTGCAGCA?CTTGAGGGTG?GGCGATTTCC?2460

TGGGGGGAGT?GGCTTAAGCC?GCTTCTCCAC?CGACCAGTTG?GCTGTAGACG?GTCCATGCTC?2520

AATGGTCCAC?CACAGATATG?AAACCACTTC?TGGAGTGAGA?GTGGCGCTCA?GTTGCTGCCC?2580

ACAGGGTGAC?TTAAATGTCC?CAAGCTGGAA?GGTGGAGAGA?GACGTGGACG?CCCCCTGGGC?2640

TCTGGGCCAC?CCTCGAGGTC?AGTAAACTAG?GAAGTTGGCT?CAAAATTAGG?AAGGGTGGGT?2700

GCCTCATGCA?GTTTGGGGGC?CCCAGGGGAG?TGAGGGCGTG?GACTTGGGAC?ACTAAAGGGC?2760

CCAAGGGTCA?AGGAGTGACC?CAAACGATGC?TGTGTGTGTG?TGTGCGTGCG?TGTGAGGGGA?2820

GGGGAGGCAT?TGTTACAAGT?TCTCTCTCCC?CACCCCCACC?TCCAGCCCCT?TCCCCACCCC?2880

CACCCGCCGG?CCTATCTCCC?CTCCCCACCC?TACACAATGG?TCTGGCCTCA?GCCCCTCCCC?2940

AGACTCCCAG?TGCTTTCCGA?TTTGGGTCCC?TTGTCTCAGA?TGCCCCCAGG?CAGCCAGCTC?3000

TCTCCCCACC?GGCCCGGGCC?TCCTTGCAGC?TCCCGCCGCT?TCCCCTCGTG?GGCTGCACTG?3060

GCATTCTCCT?TCCCTTCCAG?ACATGTGGTC?CACTAGAGCT?ACTGAACTTG?GGAACTCCCA?3120

GGTGTGACTG?ACTTGGCGGC?CTCAGACCTA?CCCTGGTGCC?CCCAGGTGTT?CCGTTGCCCT?3180

TGGTGCTGGA?AGGTTCCTTC?CCAGCCAAGG?GACTCGGCAG?ACACCAGGCA?GGCCTTATGG?3240

CCGCCCATGC?CCTCTGCGGC?CTCCTGGCCT?CTGGGGAAGA?AGCCAAGATG?GCTGGTGCTC?3300

CCTCAAGCCA?GGGGCTCAGG?CTGGGCAGCT?GGGCCAGCGT?GCCTGGGGGA?CTGGAGGGCA?3360

GGCCAGAGAA?GCATGGAGCC?AGGCTGGGTG?AGAGGCCAAC?CCATCTACCA?CAGTCTTAGC?3420

CAGTCTTCCT?GAGGGCTGAG?GCCTGTGTAG?CCTCCCTCGT?GCTTGTAGAG?CCTGGCTGGC?3480

TATGGACACT?GCAGGGCCTG?CTTTATGAGG?ACTGGTGAGA?GGAGGGGCAG?ATGAGGAGAA?3540

TGCGGGATCC?TTGGTGAGCT?CGAATACAGA?GCCAGGATGA?GGCTGGCAGG?GAAGGTGTTT?3600

GTTATAGTCT?TATCAGTGTT?GTGTCAGCAG?ATCTCAATTT?TTTCCCTTGC?CGTGCCCAGA?3660

GCCTTGGCTC?ATCCCTTTTA?AAATGCAAGG?GACAGAGGCC?AGTCCTGGGC?TCCAAGACAC?3720

TATCTGCTTG?AAGTAAGAAG?ACTCCAGGAG?GAGGAAGGTG?GGTCTTGGTG?AAGGTGGGGC?3780

TTTGAAAAGT?CTGCAGAATT?GCCTGTGTGC?ATTCATTCAC?CCCTTGGTTC?CAGGGCCCTT?3840

CGGTGCCAAG?ATGCTGGCTG?GATGCTTTCT?GGAGGTCCCG?TTTCCTCCCT?TCCAACCTCA?3900

AGCCAGGGGA?GCCTGTGTAA?TCTGGGGGGC?CTTGCCTCCT?TGGTTTCATG?CCTTTTCACT?3960

TCCCTTCCAA?TAACTGTGGG?GCCAGGGGAG?GCCAGCTGGG?CAAAGCCCCC?ATTTGAGCTC?4020

TGACGTTCTG?CCCCTTATTC?CATGAGTGCA?CCTGGTAAGC?GCCGGCTCTG?CCTTGCCTTG?4080

CGTTTCCAGG?GAGCCATCAC?GGAAAAGACT?GTTGCACCTG?AACCCCATCA?ACCAGTTGTC?4140

TACTTAAGTA?TGATTAGCTG?CCCCAGGCGG?CCTCTGGCCC?CAGACTCTGC?TTGTCCAGAC?4200

CCACCTGAGT?TGACCTGAGG?TGCCGAGCCT?CTCCACGGAG?CTACTTTTTC?TCAGCAAGCT?4260

TTTGAGGAAA?AAAAGAAGTA?GGGTATTACC?TACTCGGAAC?TTCTCTTACC?TCCTGGGGGA?4320

AGAGAGGGTG?GAGGGGGCTG?GGCCTGGCCG?GGAAGGGTAG?GTAGGGTGGG?ACAGGGGAGG?4380

GGAAGGGAGG?TGGGTCTTGC?CTGGGCAGAA?GAGTGGTGGG?TGTATCTGGA?TACTGCAGAG?4440

TGTAATCAGG?CAAGTCACCT?TTAATCGAGG?TTTCTGGACC?TCGATTTCCT?TAACTGTAAA?4500

ATGGCCAGGT?TTGTCTCAAA?GCCCCAAGAA?TCCATGTGTT?TCTGCATCCT?ATGGATGATG?4560

AGAGTTTGGG?GCCCTGTATC?TCCCTGGATG?GTGGAATTAC?AAGGGTTAGG?AGGGAAGCTC?4620

TGGAGTTGAT?TGCTGTTTTT?GTAGCTTTAG?GAGCTGTTCG?GTCTTGGGAA?TGTTATTGAA?4680

TTTAACCCCC?AAGCCTCTGA?TTTCTCAAGG?GTAAACCGGG?AGTAGCCATC?CTAGTGGCGG?4740

TGCAGGTGTG?ATGGGTTGTG?GTTCACGTGG?AGGGAAAGTC?AGAACCACAT?ATAAATAAAT?4800

AAGGTTACCT?CTGAAAGGCG?TGAAATCCAA?ATGCTCAGTG?TAAAGCCCAC?AGAAACTTGT?4860

CATCACTCAA?AGGAACATCA?TGCTCTGTTC?ACAGAGTGGA?AGGGCAGGAA?GAATCACCCA?4920

CACAATGTAA?TTGTCCCCTT?TTATTTTTTT?GAGATTTGGT?TGAATTACCT?AAGTTAAATG?4980

Real-time quantitative TaqMan round pcr is adopted to detect g.103457401A>C main effect mutational site.Utilize Primer Express3.0 software design a pair Auele Specific Primer and probe, upstream and downstream primer sequence is respectively: 5 '-GGG TAG AGT GGA CGA GAC AAT AGC-3 ' and 5 '-CCG AGA TGA AAG AAG TGC AAA TT-3 '; Probe sequence is CAT TGG AAG TGC CC (5 ' end FAM mark, 3 ' end MGB mark) and TAC CAT TGG AAG TGA CC (5 ' end VIC mark, 3 ' end are with MGB mark).Containing 30-50ng genomic dna, each 2pmol of upstream and downstream primer in 10 μ l reaction systems, two each 1pmol of probe, carry out gene type judgement after slight mixing on 7900HT FAST quantitative PCR apparatus.Pcr amplification circulation is: 50 DEG C of 2min; 95 DEG C of 10min; 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.Genotype sentences type method as shown in Figure 4 A, and arrow 1 is depicted as AA genotype individuals; Arrow 2 is depicted as AC genotype individuals; Arrow 3 is depicted as CC genotype individuals.

After adopting pcr amplification, directly the method for electrophoresis detects the genotype in g.103458678_103458679ins291 main effect mutational site.Upstream and downstream amplimer is respectively 5 '-AGG GGC AGA TGA GGA GAA TG-3 ' and 5 '-CCA AGG GGT GAA TGA ATG-3 '.In polymerase chain reaction (PCR) reaction system of 25 μ L, comprise 40ng pig genomic dna, 1.0mM MgCl ₂, 0.2mM dNTP, each 10pmol of forward and reverse primer, 2.5 unit archaeal dna polymerases (Taq enzyme) and 1 × PCR buffer (damping fluid) (company is widely collected in Shanghai).Pcr amplification condition is: 95 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations; Finally extend 10min at 72 DEG C.PCR primer, through 1.5 ℅ agarose gel electrophoresis, after ethidium bromide (EB) dyeing, observes electrophoretic band in gel imaging system.Specifically sentence type method and see Fig. 4 B.Ins/ins individuality only has the band of a 596bp, and del/del individuality only has the band of a 305bp, and ins/del individuality has the band of 596bp and 305bp respectively.

To g.103457401A>C site and g.103458678_103458679ins291 two main effect mutational sites at 1003 white duroc × Erhualian F of known vertebra number phenotypic data ₂sources group and 44 Erhualian × Tongcheng pig F ₂detect in sources group individuality, found that these two sites are in complete linkage imbalance in heredity.The cognation of application GenABEL software to genotype and phenotypic data is analyzed, and the results are shown in Table 4.As seen from the table, there is pole significant correlation in these two sites and pig rib number, g.103457401A>C the CC genotype individuals in site about 1 root bone more than AA genotype individuals; Equally, ins/ins is g.103458678_103458679ins291 individual than del/del individuality also many about 1 root bone numbers.

Second object of the present invention is achieved in that the genotyping techniques of two the main effect marks utilizing the present invention's first object to set up, and will increase by two main effect tag application of pig rib number in kind of a swine improvement.Select in nucleus herds of breeding pigs the CC genotype individuals in g.103457401A>C site or ins/ins g.103458678_103458679ins291 individual, eliminate AA genotype or the del/del genotype individuals in these two sites, by the frequency improving allele C or ins from generation to generation, increase the rib number of population with this and then reach the object increasing body length and increase meat yield.

The present invention authenticated the main effect mark that two increase pig rib number: a mononucleotide polymorphic site (SNP) and insertion/deletion (ins/del) mutational site, any one in these two sites is detected by modern molecular biology technique, utilize marker assisted selection (MAS) to select beneficial gene type individuality to reserve seed for planting, effectively can improve the rib number of population, increase individual body length and meat yield.

Accompanying drawing explanation

Below in conjunction with accompanying drawing, the present invention is described.

Fig. 1 is the schematic diagram of the GWAS positioning result No. 7 karyomit(e)s affecting pig rib number, wherein: A is white duroc × Erhualian F ₂the GWAS positioning result of sources group, B is Erhualian × Tongcheng pig F ₂the GWAS positioning result of sources group, C is the GWAS positioning result of too pig of reviving, and D is the GWAS positioning result of 3 colony's confluence analysises.18 euchromosome message identifications of pig are in X-axis, and-log10 (P) value of 60,000 SNP and rib data/coherency presses the position display of SNP in genome in Y-axis.

Fig. 2 is the schematic diagram of the Q karyomit(e) shared region No. 7 karyomit(e)s increasing pig rib number, and wherein: A is the IBD analytical results of 60K SNP chip scan-data, B for increasing the IBD analytical results after new SNP marker scanning in the IBD region shown in A.ID represents individual number; F ₀and F ₁represent the individual generation; In A and B ID be 73 " 1 " " 2 " numerically face corresponding be SNP information.Q chromosomal IBD region black surround identifies.

Fig. 3 is 4 the conservative Analysis schematic diagram of candidate main effect site g.103446231G>A, g.103451235C>T, g.103457401A>C and g.103458678_103458679ins291 in Mammals, and four grey munnions are respectively four the sequence situations of candidate main effect mutational site in 6 species; Wherein: Sus scrofa is pig, Bos taurus is ox, and Canis Lupus familiaris is dog, and Equus caballus is horse, and Homo sapiens is people, and Mus musculus is mouse, and wild is wild-type, and mutation is saltant type; " " statement is identical with pig wild-type sequence, and "-" represents vacancy.

Fig. 4 is the genotype process decision chart in g.103457401A>C and g.103458678_103458679ins291 site, wherein A be g.103457401A>C site sentence type figure, arrow 1 is depicted as AA genotype individuals; Arrow 2 is depicted as AC genotype individuals; Arrow 3 is depicted as CC genotype individuals.B be g.103458678_103458679ins291 site sentence type figure, M is 100bp molecule marker; Swimming lane 1,3,4 is del/del genotype; 2 is ins/del genotype; 5 is ins/ins genotype.

Embodiment

For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment and accompanying drawing, these embodiments only play illustrative effect, are not limited to range of application of the present invention.

Individual for nucleus herds of breeding pigs, utilize above-mentioned real-time quantitative TaqMan PCR to detect and after pcr amplification direct electrophoresis method discriminating pig No. 7 karyomit(e)s on two main effect mutational site genotype affecting rib number, favourable genotype is selected to reserve seed for planting to individuality, the rib number and the body that increase population after swarm robotic system seed selection are long, increase meat yield with this.

Embodiment

Embodiment 1:

Kind-revive too swinery body and west three way cross market pig-duroc × (Large White × landrace) market pig are laboratory animal to utilize China to cultivate pig.461 revive that too pig individual feeding is butchered to 240 ages in days, duroc × (Large White × landrace) market pig in the slaughterhouse animal-slaughtering in fixed place of Jiangxi Guo Hong company, record the rib number of each individuality respectively.Adopt above-mentioned real-time quantitative TaqMan PCR to detect and after pcr amplification directly the method for electrophoresis g.103457401A>C these 435 revive too pig and 1403 duroc × (Large White × landrace) ternarys market pig individuality of mixing is carried out and the genotype judgement of g.103458678_103458679ins291 two main effect marker sites.Then utilize Gen ABEL software to carry out the influential effect of genotype to phenotype, result is as shown in table 4.Known from this table, g.103457401A>C the CC genotype individuals in site is than AA genotype individuals on average many 1.17 (too pigs of reviving) and 0.92 root bone (west three way cross market pig), equally, ins/ins is g.103458678_103458679ins291 individual than del/del individuality also on average many 1.17 (too pigs of reviving) and 0.92 root bone (west three way cross market pig) (see table 4).It can thus be appreciated that, in business boar and Chinese native pigs core group, Systematic Breeding is the CC individuality in site or the individuality of ins/ins g.103458678_103458679ins291 g.103457401A>C, all progressively can improve the rib number of swinery, reach the object of body length and the meat yield increasing pig.

Claims

1. a main effect mark, is characterized in that: the sequence of this main effect mark is as shown in sequence table SEQ ID NO:1, and the position wherein between this sequence the 3678th and 3679 is the insertion mutation of 291 Nucleotide.

2. main effect according to claim 1 is marked at too pig of reviving, the west three way cross market pig obtained is hybridized, the application in the pig rib number qualification of the hybridized pig of the hybridized pig of duroc and Erhualian and Erhualian and Tongcheng pig by duroc, landrace and Large White.