CN101805739A - Identification method of PPARD major gene, establishment of molecular breeding method and application thereof - Google Patents

Abstract

The invention relates to an identification method of PPARD major gene, establishment of a molecular breeding method and application in the genetic improvement of breeding pigs. In the invention, by adopting the white Duroc * Erhualian Intercross and the Chinese and exotic breeding pigs of distant population, the whole-genome scan and linkage mapping, the IBD fine mapping of the target region based on a high-density SNP mark and the selection effect analysis of the distant population are carried out, and then the major gene, i.e., PPARD for deciding the traits of pig ears, fat deposition, weight of pig head and carcass traits, and the mutation site of key cause, i.e., G32E are identified. The sites are detected by the modern molecular biotechnology; by adopting the gene-assisted selection method, the beneficial gene-typed individuals are selected for the breeding pigs, thereby the breeding improvement efficiency of the traits of pig ears, fat deposition, weight of pig head and carcass traits are remarkably improved. The invention further discloses an identification course of the gene and the cause mutation site and a method for breeding the traits of pig ears, fat deposition, weight of pig head and carcass traits by using the gene marker of the invention.

Description

The discriminating of PPARD major gene, the foundation of molecular breeding method and application

Technical field

The present invention relates to the Animal Genetics field, relate to specifically differentiate that one influences pig remebrance shape, fatty deposits, body length and nose heave major gene, the foundation and the application of this gene in kind of swine improvement of molecular breeding method.

Background technology

China is the big country of raising pigs, and also is the abundantest country of pig kind resource, begins very early wild boar is domesticated for tame pig.Select through long-term, formed many local variety that differ from one another, what only " Chinese Pigs kind will " (Zhang Zhongge chief editor, Science and Technology of Shanghai press, 1986) were put down in writing just has 48.Compare with external market pig, the place of china kind has formed the bodily form appearance and the germplasm characteristic of significant difference in secular seed selection history.With remebrance shape and body length is example, and the big sagging pig kind of ear has painted face in Beijing opera, Mei Shan, eight eyebrows, the big ear pig of the river bend etc., and the pig kind of the little setting of ear has the pig of Tibetan, the southern regions of the Yunnan Province microtia pig etc.; The pig kind that build is big has painted face in Beijing opera, Mei Shan, the big ear pig of the river bend, Laiwu pig etc., and small pig kind has the pig of Tibetan, the southern regions of the Yunnan Province microtia pig, Wuzhi Mountain pig etc.Abundant germ plasm resource also provides ideal animal model (Andersson﹠amp for human medical research simultaneously not only for the genetic improvement of pig provides valuable breeding material; Georges.Nat Rev Genet.2004,5:220-212).

The described pig important economical trait of patent of the present invention relates separately to: (1) remebrance shape comprises that ear area, ear weigh and the ear type; (2) fatty deposits proterties, the average thickness of backfat during as slaughter weight; (3) nose heave; (4) trunk is long, comprises that trunk is directly long and trunk is tiltedly long.These proterties all belong to quantitative character, by controlled by multiple genes, wherein have key-gene (major gene) effect.Adopt the conventional breeding method need carry out phenotype test accurately to the seed selection of quantitative character, spend morely, selection cycle is longer.The development of Protocols in Molecular Biology, make people can seek the key-gene or the molecule marker closely linked of control pig quantitative character at dna level with it, in breeding process, be used for gene assisted Selection (gene assisted selection) or marker assisted selection (marker assisted selection), can obtain higher selection precision, select to make progress and bigger economic benefit faster.

Both at home and abroad the researchist adopts the genome scanning method, and (SSC7) located utmost point remarkably influenced pig remebrance shape, fatty deposits, nose heave and quantitative trait locus (QTL) that trunk is long on No. 7 karyomit(e)s of pig.Wei etc. are material with Mei Shan * Da Bai three generations family, and the locus that utilizes 152 marks will influence ear area and ear type is positioned in the SSC7 58cM adjacent domain, fiducial interval less than 20cM (Wei et al.AnimalGenetic 2007,38:222-226).It is that the main effect QTL that maternal a plurality of resource familys will influence the fatty deposits proterties also is positioned SSC7 58cM adjacent domain (Sanchez etal.J Anim Sci.2006,84:526-537 that a plurality of research groups utilize with Mei Shan; Bidanel et al.Genet Sel Evol.2001,33:289-309; Rattink et al.Mamm Genome. 2000,11:656-661).Edwards etc. (J Anim Sci.86:254-266) utilize 124 microsatellite markers to Du Luoke * Pietrain pigs F ₂Resource colony carries out genome scanning, finds to have the main effect QTL that influences body length on No. 7 karyomit(e)s.(Science in China.2003,46:10-17) three F that constitute with wild boar, plum mountain pig and Pietrain such as Yue ₂Colony is a research object, has found that at SSC7 several influence the QTL of proterties such as body is long, nose heave, the intercostal thickness of backfat of 13-14, shoulder fat thickness.

The applicant is from calendar year 2001 to 2006 year, utilize the time in 6 years to make up large-scale 4 a platinites Du Luoke * painted face in Beijing opera resource colony, in this colony, measured the phenotype index of 422 of 7 big classes (apparent, production, trunk, meat, breeding, healthy relevant and behavioral trait), the F of the remebrance shape wherein arranged, trunk is long, nose heave and the fatty deposits phenotype writes down ₂Individual above 1000, F ₃Individual 560.Because first ancestor white Du Luoke is the lean meat species male parent line that is widely used in commercial production, and the painted face in Beijing opera pig is many and be easy to fatty deposits and well-known Chinese native pig breed with litter size, therefore at F ₂And F ₃There is the obvious phenotypes separation phenomenon in the said determination proterties in the colony.The applicant utilizes 194 microsatellite markers that white Du Luoke * painted face in Beijing opera resource family has been carried out full genome scanning and QTL positioning analysis, a utmost point remarkably influenced remebrance shape, fatty deposits, trunk length and nose heave QTL have been found at SSC7 58cM place, the QTL that wherein influences the ear area is in this resource colony detected 1, among 264 QTL the most significant one, fiducial interval only is 2cM, soluble surpass 40% phenotypic variation (Ma et al.Anim Genet.2009,40:463-467).Noticeablely be: the QTL genotype of painted face in Beijing opera has increases ear weight, ear area, body is long, nose heave and the effect of minimizing fatty deposits.The applicant is on this working foundation, carried out the hereditary analysis research of this QTL in a deep going way, the major gene (QTG) and cause and effect mutational site (QTN) that influence this QTL have finally been differentiated, not only disclose the molecular genetic mechanism of these phenotypic characters thus, also set up the novel molecular breeding technique be used for boar remebrance shape, fatty deposits, the nose heave and long seed selection improvement of trunk accordingly.

Summary of the invention

First purpose of the present invention provides one influences pig remebrance shape, fatty deposits, nose heave and major gene that trunk is long.

Second purpose of the present invention provides a kind of molecular breeding method at pig remebrance shape, fatty deposits, the nose heave and long proterties of trunk.

The 3rd purpose of the present invention is the application of major gene in kind of swine improvement that influences pig remebrance shape, fatty deposits, the nose heave and long proterties of trunk.

First purpose of the present invention is achieved in that

One influences pig remebrance shape, fatty deposits, nose heave and major gene that trunk is long, and feature is: the cDNA sequence of this gene has one of following nucleotide sequence:

(1) polynucleotide of SEQ ID No:2 in the sequence table, it is by 2717 based compositions, the encoding sequence of this cDNA sequence be from the 102nd at 5 ' end to 1427 bit bases, its sequence is as follows:

gttgacagga?actgccctgt?agaggtccat?ctgcactcag?acccagatga?tgccagagct 60

atgaccgggc?ctgcaggcgt?ggcgccgagg?ggaagtcagc?catggagcag?ccgccggagg 120

aagcccctga?ggtccgggaa?gaggagaaga?aaaaggaagt?ggcagaggcc?gaaggaggcc 180

cagagctcaa?tgggggacca?gagcactcgc?ttccctccag?cagctgtaca?gatctctccc 240

agagctgctc?tccacccgcg?ctgctggacc?agctgcagat?gggctgcgac?ggggcctcgt 300

gcggtggcct?cagcatggag?tgccgggtgt?gcggggacaa?ggcatcaggc?ttccactacg 360

gagtccacgc?ttgcgagggg?tgcaagggct?tcttccgccg?gacaatccgc?atgaagctgg 420

agtacgagaa?gtgtgagcgg?atctgcaaga?tccagaagaa?gaaccgcaac?aagtgccagt 480

actgccgctt?ccagaaatgc?ctggcgctgg?gcatgtctca?caacgccatt?cgctttggcc 540

ggatgcccga?ggcagagaaa?aggaagctgg?tggctgggct?gacggcaaac?gaggggagtc 600

agcacaaccc?gcaggtggct?gacctgaagg?ccttctccaa?gcacctctac?agcgcctacc 660

tgaaaaactt?caacatgacc?aaaaagaagg?cccgcgccat?cctcaccggc?aaggccagcc 720

acacagcgcc?ctttgtgatc?cacgacatcg?agacgttgtg?gcaggccgag?aagggcctgg 780

tgtggaagca?gctggtgaat?ggcctgccgc?cctacaagga?gatcagcgtg?cacgtcttct 840

accgctgcca?gtgcaccacg?gtggagacgg?tgcgcgagct?gaccgagttc?gccaagagca 900

tccccagctt?cgaccacctc?ttcctcaacg?accaggtgac?ccttctcaag?tacggcgtgc 960

acgaggccat?cttcgccatg?ctggcctcca?tcgtcaataa?ggatgggctg?ctggtggcca 1020

acggcactgg?ttttgtcacc?cgcgagttcc?tgcgcagcat?ccgaaagccc?ttcagtgaca 1080

tcattgagcc?caagtttgag?ttcgctgtca?agttcaatgc?cctggaactc?gatgatagtg 1140

acctggctct?cttcatcgca?gccatcattc?tgtgtggaga?ccggccaggc?ctcatgaacg 1200

tgtcacaggt?ggaggccatc?caggacacca?tcctgcgtgc?cctcgagttc?cacctgcagg 1260

ccaaccaccc?cgacgcccag?tacctcttcc?ccaagctgct?gcagaagatg?gcagacctgc 1320

ggcagctggt?caccgagcac?gcccagatga?tgcagcggat?caagaagacc?gagaccgaga 1380

cctcgctgca?ccccctgctc?caggagatct?acaaggacat?gtactgaggg?gtgcgccttg 1440

ggcctcccaa?caggcctccc?ggagcaggtg?gacggcgcgg?ggacagacac?tgcctgcggg 1500

acgtttccgt?tgaccagccc?gagccctcag?ccgagcagca?ggtcacaggc?tcagccagac 1560

gcacggcctc?ccactcctta?tagccctgcc?tcctctccct?cctcagctcc?cctctctctc 1620

atctctttgc?tctttctttt?ccttcctctc?tcagcctcgc?tttctctctc?cccatcctgt 1680

ctgtccatct?ttctcttcct?gtgagacagt?ttgtgttatt?tcaccagcac?caaaacaaga 1740

ccgctgcttt?gtcccctgct?ccccggcccc?ggagcaggag?ggggcagggc?ctgccctctg 1800

caccaaccat?cgccttctcc?agtcttcaaa?ggacacgcag?gccatccaaa?gaaacactaa 1860

gctctccggg?cctggcttac?tggggaagcc?acgcagggcc?tgggctgagt?gccgagcagc 1920

cctagccaca?gggtccctgg?gggaggccgc?ccacccgagg?ctgaggctgg?caccccatgg 1980

ctgagcggac?cccgctcctg?cagcatgcct?cagccccaca?gacgcccacc?cctcttttgt 2040

ttttctttgc?accagtcttc?caggccagtg?ccactgtgct?ggctgctggc?ggatgccccc 2100

agcctggatg?gaggtgggat?tccctccagg?tgggggcgcc?cacaccccca?ttgaagagga 2160

gcatgcctca?agggagcagt?tggtagggaa?ggcagtgggc?agcagacttg?attctgaccc 2220

caggccttgg?gtgggtcctc?cctcagcacc?ccactctctc?cagcctctgc?agcagccact 2280

gagccctgcc?cacgctgtgt?cagcatcgca?cctcccacct?ccgcagcacc?ccggcttggc 2340

ctcagccacg?cccttctttc?tccagccggg?cgacactggc?tccagcccag?ctgaagcgca 2400

cactccctgg?agcgcctcca?gcacacgcag?cacgagcact?gaaatcactt?tacctgcagg 2460

ttccacaacc?tcggcctccc?tcctgaggca?ggtggaccac?agagctgtgc?ccctgactcc 2520

ccgggcgggc?ggggagccct?gctgccccag?cccagcactg?ctcgcagggg?aggtacccag 2580

gatgaactga?tcccgctcac?ttgtgacacc?catttgttcc?agcagctctg?ctgccctccc 2640

ctttccttgt?gattggccca?gccaggcacc?tggagctctc?cctgcaccgc?ttctggtgac 2700

cagggaccct?gccaggc 2717；

(2) amino acid of SEQ ID No:1 in the code sequence tabulation, it is made up of 441 amino-acid residues, and its sequence is as follows:

MEQPPEEAPE?VREEEKKKEV?AEAEGGPELN?GEPEHSLPSS?SCTDLSQSCS?PPALLDQLQM 60

GCDGASCGGL?SMECRVCGDK?ASGFHYGVHA?CEGCKGFFRR?TIRMKLEYEK?CERICKIQKK 120

NRNKCQYCRF?QKCLALGMSH?NAIRFGRMPE?AEKRKLVAGL?TANEGSQHNP?QVADLKAFSK 180

HLYSAYLKNF?NMTKKKARAI?LTGKASHTAP?FVIHDIETLW?QAEKGLVWKQ?LVNGLPPYKE 240

ISVHVFYRCQ?CTTVETVREL?TEFAKSIPSF?DHFFLNDQVT?LLKYGVHEAI?FAMLASIVNK 300

DGLLVANGTG?FVTREFLRSI?RKPFSDIIEP?KFEFAVKFNA?LELDDSDLAL?FIAAIILCGD 360

RPGLMNVSQV?EAIQDTILRA?LEFHLQANHP?DAQYLFPKLL?QKMADLRQLV?TEHAQMMQRI 420

KKTETETSLH?PLLQEIYKDM?Y 441；

(3) with sequence table in the nucleotide sequence that limits of SEQ ID No:2 have 90% above homology, and the proteinic nucleotide sequence of identical function of encoding;

(4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID No:2 in the sequence table.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

Above-mentionedly influence pig remebrance shape, fatty deposits, the coded proteinic name of major gene nose heave and that trunk is long is called peroxisome proliferation-activated receptors D (PeroxisomeProliferator-Activated Receptor Delta, PPARD), derive from pig, amino acid residue sequence with SEQ ID No:1 in the sequence table, through replacement, disappearance or the interpolation of 1-10 amino-acid residue and with pig remebrance shape, fatty deposits, nose heave and trunk grow proterties relevant by SEQ ID No:1 deutero-protein.

Second purpose of the present invention is achieved in that

Foundation is at the molecular breeding method of boar remebrance shape, fatty deposits, the nose heave and long proterties of trunk:

(1) nucleotide sequence of SEQ ID No:3 and SEQ ID No:4 in the use sequence table, pig genomic dna to be measured is carried out pcr amplification, dyeing is carried out single nucleotide polymorphism to pcr amplification product and is detected, and determines that from 5 ' end the 565th bit base of SEQ ID No:3 or 5 ' end the 121st bit base of SEQ ID No:4 be G or A;

(2) if the mononucleotide polymorphic result of pcr amplification product holds the 121st bit base (promptly holding the 565th bit base from 5 ' of SEQ ID No:3) to be G from 5 ' of SEQ ID No:4, its homozygotic genotype is GG; 5 ' end the 121st bit base (promptly holding the 565th bit base from 5 ' of SEQ ID No:3) from SEQ ID No:4 is A, and its homozygotic genotype is AA; Their heterozygote genotype is GA; The individual ear area of AA genotype, ear weigh, trunk is long and the nose heave utmost point is higher than GA genotype and GG genotype individuality significantly, significantly are being lower than GA genotype and GG genotype individuality aspect the fatty deposits proterties (the average thickness of backfat).

Below by experiment first purpose of the present invention and second purpose are verified:

1. utilize white Du Luoke * painted face in Beijing opera F ₂Resource family Fine Mapping goal gene:

The applicant has increased by 17 SNP and 11 microsatellite markers of 8 genes in the SSC7 QTL interval of having reported early stage, detected white Du Luoke * painted face in Beijing opera resource family F ₀, F ₁And F ₂For whole individual genotype at these marker sites.According to Nezer etc. (Genetics.2003,165, mark 277-285) is assisted partition method, utilizes the information of all 32 marks in the QTL interval, infers F ₁And F ₂The QTL genotype of boar, result show 9 F ₁Boar all is Qq genotype (accompanying drawing 1).All 9 increase ear area and the heavy shared one section 1.4Mb chromosomal region (SW0671 SRPKI) of Q karyomit(e) of ear, and find to share haplotype (accompanying drawing 2) in q karyomit(e).The above results shows that fully goal gene (QTG) is positioned at determined 1.4Mb chromosomal region.

In view of the extremely significantly remebrance shape phenotypic difference that is shown between painted face in Beijing opera and white Du Luoke kind, the applicant infers painted face in Beijing opera and white Du Luoke has fixed Q and q allelotrope respectively, and all for generations the painted face in Beijing opera sow all carry Q karyomit(e).In order to verify this hypothesis, the applicant has made up 19 haplotypes of individual (2 boars and 17 sows) for generations, find the haplotype of 34 karyomit(e)s shared one section 1.0Mb in SW0671 SRPKI1.4Mb zone of all painted face in Beijing opera first ancestors, this haplotype has extremely significantly increases ear area and the heavy effect (accompanying drawing 3) of ear.Based on this result, we are with the chromosomal region of goal gene site Fine Mapping to HMGA1 to 1.0Mb between the NC3652.

2. utilize the further Fine Mapping goal gene of selection effect of edge far away colony:

Ear is sagging greatly to be one of varietal characteristic of painted face in Beijing opera pig.In secular seed selection history, the painted face in Beijing opera of big ear type is preferentially selected and remain.Farmer's proverb cloud: " big person of ear such as Buddhist, the little person of ear such as thief " (Zhang Zhongge chief editor, Chinese Pigs kind will, Science and Technology of Shanghai press, 1986).The applicant infers influences the QTG zone of remebrance shape at this, and the painted face in Beijing opera pig is owing to be subjected to the influence of selection effect can produce the phenomenon (selectionsweep) that heritable variation significantly reduces.In order to verify this hypothesis, the applicant has collected 119 no relevant individual sample of 216 samples and 3 the west commercial varieties of 211 samples of 3 core groups representing all painted face in Beijing opera genetic backgrounds, 6 place of china pigs.Utilize that 7 little satellites and 31 SNP marks detect above-mentioned sample in this 1.0Mb zone of HMGA1-NC3652, the heritable variation degree of finding painted face in Beijing opera pig 22 adjacent markers in the 680kb zone of PACSIN-FANCE extremely significantly descends, and the high frequency gene frequency of nearly all mark has all surpassed 0.90.Especially what deserves to be mentioned is, these 22 be marked at all do not have in 72 Xishan painted face in Beijing opera pigs polymorphic.By contrast, these are marked in other Chinese native pig breeds, the commercial pig kind in west and the wild boar all higher polymorphism (accompanying drawing 4).This shows that the painted face in Beijing opera pig lives through the intensive forward and selects in this 680kb zone, and tangible selection effect is arranged, and can infer the chromosomal region that goal gene is positioned at this 680kb thus.

3. the discriminating of goal gene (QTG):

Above-mentioned 680kb interval comprises 13 functional genes of note corresponding to people's homologous region.The applicant has detected the expression of these 13 genes in ear tissue by sxemiquantitative inverse transcription polymerase chain reaction (RT-PCR), find PPARD, FANCE, TAFII, 5 genes such as ZNF76 and SNRPC are expressed at the ear tissue camber, and other expression of gene amounts are very low or do not express.In the gene that 5 high abundances are expressed, FANCE, TAF11, ZNF76 and SNRPC are house-keeping genes, they are that the possibility of goal gene is little.PPARD is the transcription factor of part regulation and control, belongs to nuclear receptor superfamily, in a plurality of significant process of biology, play a crucial role (Barish et al.J Clin Invest.2006,116:590-597).For example, PPARD facilitates cPLA ₂Key molecule (the Han et al.J Cell Biochem.2008 of α activation β-catenin and then regulation and control Wnt/ β-catenin path, 105,534-545), this path in various kinds of cell behaviors such as chondrocyte proliferation and differentiation, bring into play keying action (Macsai et al.J Cell Physiol.2007,215:578-587).In addition, PPARD still is the crucial regulatory factor in the metabolism of fat, and it promotes adipocyte and myocyte's fat combustion, increase energy expenditure, and this makes this gene become research focus (the Wang et al.Cell.2003 in obesity and the treating correlative diseases, 113,159-170; Evans et al.Nat Med.2004,10,355-361).Learn function and the effect in cartilage development and metabolism of fat process thereof in view of the known organism of PPARD, PPARD is a most possible goal gene in this QTL zone.The applicant has detected the PPARD gene 0 by real-time reverse transcription PCR subsequently, 45,90, expression in the painted face in Beijing opera of 300 ages in days and the duroc ear tissue, find that these two interracial PPARD gene expression amounts do not have significant difference (accompanying drawing 5), inference is likely the structural mutation of PPARD rather than regulates the hereditary effect that sudden change has caused this QTL thus.

4. the discriminating in crucial cause and effect mutational site (QTN):

The applicant searches the CH242-397F5 BAC clone (position is 35255108-35468260) who contains pig PPARD gene on ENSEMBLE website (http://www.ensembl.org/index.html).(http://www.ncbi.nlm.nih.gov/) downloads to (sequential reception number: NM_214152) of the cDNA encoding sequence of pig PPARD gene of 1826bp and part 3 ', 5 '-UTR sequence from the NCBI website.Intron, exon and the coding region of PPARD have been determined by the comparison of Powerblast software.Utilize above-mentioned from including of ENSEMBLE website (http://www.ensembl.org/index.html) dna sequence dna of one section 72kb of pig PPARD gene, use the many genomic dnas (thymus nucleic acid) individual for generations with 17 painted faces in Beijing opera for generations of online Primer 3.0 softwares (http://frodo.wi.mit.edu/primer3/) design to 2 white Du Luoke of primer amplification.Amplified fragments adopts QIAquick PCR product purification test kit (QIAGEN, Germany) purifying.By sequencing analysis relatively find primer 1 (F:5 '-TCC TGC CTG TCT TCT CGT GTC-3 '; R:5 '-CCG GCT CTTCCT GAG GTC TGT-3 ') there is mononucleotide polymorphic site (SNPs) that a guanylic acid/adenylic acid (AMP) replaces in the 565th Nucleotide place of Kuo Zeng 1066 Nucleotide composition sequences (SEQ ID No:3), is SNPG565A.This sudden change causes the 32nd amino acid of coded PPARD albumen to be mutated into Glu (G32E) by Gly.This mutational site occurs in the Mammals high conservative and has the Intrinscially disorder domain (functional domain) that important biomolecule is learned function, and this site is Gly in Mammalss such as people, mouse, ox, sheep.

The applicant has further detected the PPARDG32E genotype of all individualities surplus 1000 of white Du Luoke * painted face in Beijing opera family, by standard association, mark additional related analyze and F-descend check (Zhao etc., 2003) analyzed PPARD G32E for remebrance shape, fatty deposits, trunk is long and nose heave influences effect.Heavy (P=5.57 * 10 of standard association analysis revealed PPARD G32E and ear ^-183), ear area (P=1.04 * 10 ^-164), the average thickness of backfat (P=3.69 * 10 ^-158), nose heave (P=6.68 * 10 ^-134) and long (P=3.46 * 10 of trunk ^-134) etc. proterties all be utmost point significant correlation.The auxiliary association analysis of mark shows that also PPARDG32E is for above-mentioned affect trait remarkable (P＜0.001).In addition, in the QTL model PPARD G32E as fixed effect after, the QTL effect disappears substantially.These results have proved conclusively PPARDG32E and have been not only the crucial cause and effect mutational site (QTN) that SSC7 goes up influence the remebrance shape, and also having extremely significantly to proterties such as fatty deposits, nose heave and trunk are long, " one because of multiple-effect " influences effect.

In order to determine sudden change origin and the population genetics rule in different pig kinds of PPARD G32E, the applicant to from 10 Chinese native pig breeds, 11 European pig kinds, 3 west commercial varieties and in, 753 individualities of Europe wild boar have carried out PPARD G32E genotype and have judged.In Chinese native pig breed, increase the heavy A allelotrope high frequency (＞0.80) of ear area and ear and be present in the big sagging kind of ear, be present in intermediate frequency in the kind of medium-sized ear.On the contrary, reduce the heavy G allelotrope of ear area and ear in the Chinese native pig breed of all wild boars, European local pig, the commercial pig kind in west and small-sized ear fixing (table 1).It is the GA heterozygote that the black greatly pig in 2 Europe is arranged.This kind ear is big and vertical, and Documentary Records shows the blood relationship (http://www.ansi.okstate.edu/breeds/swine/largeblack/index.htm) that has imported the place of china pig in this kind of 19 th century later.These results show that further PPARD G32E is the major gene that influences the remebrance shape.

The gene frequency of table 1 PPARD G32E in China and foreign countries' pig kind distributes

The present invention is by detecting the single nucleotide polymorphism of PPARD G32E, can determine remebrance shape, fatty deposits, nose heave and favourable allelotype that trunk is long, select favourable genotype individuality to reserve seed for planting, can significantly improve boar remebrance shape, fatty deposits, seed selection improvement efficient and character inheritance nose heave and the long proterties of trunk are stable.

Description of drawings

Fig. 1 is white Du Luoke * 9 F of painted face in Beijing opera family that determine by the auxiliary compartment analysis of mark ₁The QTL genotype of boar; Ordinate zou is represented the auris dextra area, and X-coordinate is represented F ₁The boar family.The X-coordinate upper and lower is indicated boar overbit and corresponding Z value respectively.Every boar offspring ordered series of numbers is in the standard error top.Calculate Z value, i.e. log H according to methods such as Nezer ₁/ H ₀, H wherein ₁Suppose that boar QTL genotype is Qq, H ₀Suppose that boar QTL genotype is QQ or qq.The Q allelotype that increases the ear area represents that with rhombus the q allelotype is represented with circle.

Fig. 2 is that 9 F1 boar Q karyomit(e)s are at the interval 1.4Mb haplotype of sharing (show look square frame) of QTL; Haplotype is by the SimWalk2 software building.Microsatellite marker represents that with black italic the allelotype fragment is from being short to the long digital serial number of using, and the genotype of SNP is represented with 1 (high frequency) or 2 (low frequencies).QTL fiducial interval flank microsatellite marker (S1856 and S0666) marks with red.F ₁The boar overbit is listed in the coordinate axis left side, and every karyomit(e) QTL genotype is listed in the right side.About 1.4Mb haplotype that Q karyomit(e) is shared indicates with red block.

Fig. 3 is that individual 34 karyomit(e)s of painted face in Beijing opera first ancestor are at the interval 1.0Mb haplotype of sharing (show look square frame) of QTL;

The heritable variation degree of Fig. 4 China and foreign countries' pig kind 38 marker sites in the QTL interval; The sample size of 13 China and foreign countries' pig kinds and the heterozygosity of 38 marks as shown in the figure, microsatellite marker is represented with black italic.The result show the painted face in Beijing opera pig between PACSIN and FANCE in the 680-kb zone nearly all marker site be tending towards fixing, show this interval painted face in Beijing opera pig extremely strong selection drift (selection sweep) taken place.

PPARD gene the expression in painted face in Beijing opera and Du Luoke ear tissue of Fig. 5 for obtaining by the analysis of real-time quantitative reverse transcription PCR; 0,45 ± 3,90 ± 3 and 300 ± 3 painted face in Beijing opera and the extraction that duroc ear tissue sample is used for RNA, wherein 6 samples of each age in days of totally 4 ages in days have been gathered respectively.The real-time quantitative reverse transcription PCR is done reaction repeated three times, averages as final value.The result shows between painted face in Beijing opera and the duroc does not have significant difference at the PPARD of these 4 age in days sections expression level.

Fig. 6 is the sequence peak figure in the pig PPARD G32E point mutation site of the present invention's discriminating;

Fig. 7 declares type figure for the SNAPshot of PPARD G32E, and wherein a is the GG type; B is the AA type; C is the AG type.

Embodiment

Embodiment 1:PPARD G32E in white Du Luoke * painted face in Beijing opera resource colony to remebrance shape, fatty deposits, nose heave and trunk is long influences effect.

(1) genotype of PPARD G32E is judged:

The design primer to F2 and R2 (F2:5 '-CGG CTG TTT TAC AGG AAG GA-3 '; R2:5 '-CTG CAC TCA GAC CCA GAT GA-3 ') amplification comprises the dna fragmentation of PPARD G32E, and synthetic another of design is used for the extension primer (5 '-TTT TTT TTT TGC TGG AGGGAA GCG AGT GCT CTG GT-3 ') of SNAPshot reaction.Utilize in the PCR reaction system of primer to 15 μ l of F2 and R2 amplification pig genomic dna, comprise 40ng pig genomic dna, 35mM MgCl ₂, 20mM dNTP, each 2pmol of forward and reverse primer, 2 Taq of unit enzymes and 1 * PCR buffer (Shanghai bio-engineering corporation, China).Amplification condition is: 94 ℃ of 3min; 94 ℃ of 30s, 67 ℃ of 30s, 72 ℃ of 40s, totally 30 circulations; Extend 10min at 72 ℃ at last.The PCR product detects with 2% agarose gel electrophoresis and takes pictures.Get 2.5 μ l PCR products, add 0.5 μ l ExoSAP-IT enzyme (Shanghai traditional Chinese medicines group company, China) and hatch 45min for 37 ℃, handle the 15min inactivator for 80 ℃ then.The SNAPshot reaction system is: SNAPshot reaction mixture (Multiplex Ready Reaction Mix) 2 μ l; SNAPshot extends primer 0.2 μ l (the primer final concentration is 0.4 μ mol/L); Above-mentioned purified pcr product 2.0 μ l; Deionized water 0.7 μ l.The SNAPshot loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 30s, totally 30 circulations.Get that 5 μ l SNAPshot reaction product add 0.57 μ l 10 * bufffer and 0.1 μ l CIP (SNAPshot reaction product purifying enzyme) is hatched 1h under 37 ℃, handle the 15min inactivators and reach the product purification effect for 75 ℃ then ,-20 ℃ of preservations are standby.Get above-mentioned SNAPshot reaction product, 8.0 μ l methane amide denaturing agents (Hi-Di formamide) and the 0.25 μ l GeneScan 120LIZ size standard (electrophoresis intramolecularly mark) after purified of 1.75 μ l.System is mixed the back in 95 ℃ of sex change 5min, places immediately then and handles 2min on ice, and the centrifugal ABI of being splined on 3130XL genetic analyzer carries out capillary electrophoresis separation and genotype is judged.Specifically declare the type method as shown in Figure 2.

2.PPARD G32E is to remebrance shape, fatty deposits, nose heave and trunk is long influences effect:

With white Du Luoke * painted face in Beijing opera resource family 1000 remainder F ₂Generation is individual carries out the correlation analysis that PPARD G32E and pig remebrance shape, fatty deposits, nose heave and trunk are grown proterties for experimental subjects, and concrete grammar is as follows:

According to the method for above-mentioned steps 1, respectively with 1000 bull F ₂Genomic total DNA is a template for the pig ear tissue of pig, carry out pcr amplification at primer under to the guiding of F2 and R2, utilize the SNAPshot of ABI company technology for detection 5 '-TTT TTT TTT TGC TGG AGG GAA GCG AGT GCT CTG GT-3 ' to extend the base of back 3 ' end.Show 5 ' end the 121st bit base from SEQ ID No:4 as detected result, promptly 5 ' of SEQ ID No:3 end 565 bit bases are when being G in the sequence table, and its homozygotic genotype is GG; From 5 ' end the 121st bit base of SEQ ID NO:4, when promptly 5 ' of sequence 3 end the 565th bit base was A in the sequence table, its homozygotic genotype was AA; Their heterozygote genotype is GA.

Analyze and F-decline check analysis PPARD G32E and remebrance shape (ear area and ear are heavy) dependency of fatty deposits proterties (the average thickness of backfat), trunk length and nose heave proterties with standard association, mark additional related.Heavy (P=5.57 * 10 of standard association analysis revealed PPARDG32E and ear ^-183), ear area (P=1.04 * 10 ^-164), the average thickness of backfat (P=3.69 * 10 ^-158), nose heave (P=6.68 * 10 ^-134) and long (P=3.46 * 10 of trunk ^-134) utmost point significant correlation.The auxiliary association analysis of mark shows that also PPARDG32E is for above-mentioned affect trait remarkable (P＜0.001).In addition, in the QTL model this SNP as fixed effect after, the QTL effect disappears substantially, the QTL effect (table 2) greater than 97% can be explained in this SNP site.These results all confirmed PPARD G32E be influence the remebrance shape on No. 7 karyomit(e)s of pig, nose heave, body is long and the main mutational site of imitating of fatty deposits.The ear area of AA genotype individuality, ear weigh, trunk is long and the nose heave utmost point is higher than GA and GG genotype individuality significantly, and are markedly inferior to GA and GG genotype individuality at the utmost point aspect the fatty deposits proterties (the average thickness of backfat).By the individuality of colony's subculture seed selection different genotype, can be to the ear area of boar, ear is heavy, trunk is long, nose heave and the fatty deposits proterties obtains seed selection improved effect efficiently.

Table 2 PPARDG32E is to white Du Luoke * painted face in Beijing opera resource family F ₂Colony's purpose proterties influence effect

^aCalculation of correlation is according to reports (2003) such as Zhao.We carry out correlation detection with 4 models: y=μ+SNP+ fixed effect+concomitant variable+y=μ+SNP+ fixed effect+concomitant variable+e (model 1, SNP model); Y=μ+fixed effect+concomitant variable+e (model 2, brief model); Y=μ+QTL+SNP+ fixed effect+concomitant variable+e (model 3, QTL+SNP model); Y=μ+QTL+ fixed effect+concomitant variable+e (model 4, QTL model); Y is a character value, and μ is a mean value, and e represents residual error.In the model fixed effect be sex and batch, concomitant variable is a carcass weight.The relevant F ratio by model 1 and model 2 sum of squares of residues of standard calculates; The mark additional related is calculated by the F ratio of model 3 and model 4 sum of squares of residues; Descend the upcheck F ratio of model 1 and model 3 sum of squares of residues of F calculates.

Embodiment 2:PPARD G32E in 6 edge far away colonies to the effect that influences of ear area.

The applicant has gathered the ear tissue sample of 574 above sows of multiparity 2 tires in the colony such as edge far away such as 6 of painted face in Beijing opera pig, Hangzhoupro pig, the black pig in Yushan, Su Zhong pig, Soviet Union ginger pig and Su Tai pigs etc., extracts genomic dna.Carried out the measurement of ear area to trying the following method of individual employing: with a standard ruler and the parallel placement of pig ear, take high-resolution pig ear photo by vertical focusing, with the standard ruler is reference, utilizes LaicaQwin area measurement computed in software to go out each individual pig ear area.

Method according to step 1 among the embodiment 1, genomic dna with above-mentioned edge far away colony individuality is a template, carry out pcr amplification at primer under to the guiding of F2 and R2, utilize the SNAPshot of ABI company technology for detection 5 '-TTT TTT TTT TGC TGG AGG GAA GCG AGT GCT CTG GT-3 ' to extend the base (being amplified production 121bp place base) of back 3 ' end.Show 5 ' end the 121st bit base from SEQ ID No:4 as detected result, promptly 5 ' of SEQ ID No:3 end 565 bit bases are when being G in the sequence table, and its homozygotic genotype is GG; From 5 ' end the 121st bit base of SEQ ID NO:4, when promptly 5 ' of sequence 3 end the 565th bit base was A in the sequence table, its homozygotic genotype was AA; Their heterozygote genotype is GA.

Utilize the GLM program of SAS9.0 software to carry out the standard association analysis of PPARD G32E and ear area.The result shows the equal significant correlation of ear area proterties (P＜0.05) of PPARD G32E and each colony, and the ear area of AA genotype individuality is higher than GA and GG genotype individuality (table 3) significantly.These results have reconfirmed that PPARD G32E is the crucial cause and effect mutational site (QTN) that influences pig ear area.

Table 3 PPARD G32E in 6 edge far away colonies to the effect that influences of ear area

Claims (5)

1. one influences pig remebrance shape, fatty deposits, nose heave and major gene that trunk is long, and it is characterized in that: the cDNA sequence has one of following nucleotide sequence:

(1) polynucleotide of SEQ ID No:2 in the sequence table, it is by 2717 based compositions, and its sequence is as follows:

gttgacagga?actgccctgt?agaggtccat?ctgcactcag?acccagatga?tgccagagct 60

atgaccgggc?ctgcaggcgt?ggcgccgagg?ggaagtcagc?catggagcag?ccgccggagg 120

aagcccctga?ggtccgggaa?gaggagaaga?aaaaggaagt?ggcagaggcc?gaaggaggcc 180

cagagctcaa?tgggggacca?gagcactcgc?ttccctccag?cagctgtaca?gatctctccc 240

agagctgctc?tccacccgcg?ctgctggacc?agctgcagat?gggctgcgac?ggggcctcgt 300

gcggtggcct?cagcatggag?tgccgggtgt?gcggggacaa?ggcatcaggc?ttccactacg 360

gagtccacgc?ttgcgagggg?tgcaagggct?tcttccgccg?gacaatccgc?atgaagctgg 420

agtacgagaa?gtgtgagcgg?atctgcaaga?tccagaagaa?gaaccgcaac?aagtgccagt 480

actgccgctt?ccagaaatgc?ctggcgctgg?gcatgtctca?caacgccatt?cgctttggcc 540

ggatgcccga?ggcagagaaa?aggaagctgg?tggctgggct?gacggcaaac?gaggggagtc 600

agcacaaccc?gcaggtggct?gacctgaagg?ccttctccaa?gcacctctac?agcgcctacc 660

tgaaaaactt?caacatgacc?aaaaagaagg?cccgcgccat?cctcaccggc?aaggccagcc 720

acacagcgcc?ctttgtgatc?cacgacatcg?agacgttgtg?gcaggccgag?aagggcctgg 780

tgtggaagca?gctggtgaat?ggcctgccgc?cctacaagga?gatcagcgtg?cacgtcttct 840

accgctgcca?gtgcaccacg?gtggagacgg?tgcgcgagct?gaccgagttc?gccaagagca 900

tccccagctt?cgaccacctc?ttcctcaacg?accaggtgac?ccttctcaag?tacggcgtgc 960

acgaggccat?cttcgccatg?ctggcctcca?tcgtcaataa?ggatgggctg?ctggtggcca 1020

acggcactgg?ttttgtcacc?cgcgagttcc?tgcgcagcat?ccgaaagccc?ttcagtgaca 1080

tcattgagcc?caagtttgag?ttcgctgtca?agttcaatgc?cctggaactc?gatgatagtg 1140

acctggctct?cttcatcgca?gccatcattc?tgtgtggaga?ccggccaggc?ctcatgaacg 1200

tgtcacaggt?ggaggccatc?caggacacca?tcctgcgtgc?cctcgagttc?cacctgcagg 1260

ccaaccaccc?cgacgcccag?tacctcttcc?ccaagctgct?gcagaagatg?gcagacctgc 1320

ggcagctggt?caccgagcac?gcccagatga?tgcagcggat?caagaagacc?gagaccgaga 1380

cctcgctgca?ccccctgctc?caggagatct?acaaggacat?gtactgaggg?gtgcgccttg 1440

ggcctcccaa?caggcctccc?ggagcaggtg?gacggcgcgg?ggacagacac?tgcctgcggg 1500

acgtttccgt?tgaccagccc?gagccctcag?ccgagcagca?ggtcacaggc?tcagccagac 1560

gcacggcctc?ccactcctta?tagccctgcc?tcctctccct?cctcagctcc?cctctctctc 1620

atctctttgc?tctttctttt?ccttcctctc?tcagcctcgc?tttctctctc?cccatcctgt 1680

ctgtccatct?ttctcttcct?gtgagacagt?ttgtgttatt?tcaccagcac?caaaacaaga 1740

ccgctgcttt?gtcccctgct?ccccggcccc?ggagcaggag?ggggcagggc?ctgccctctg 1800

caccaaccat?cgccttctcc?agtcttcaaa?ggacacgcag?gccatccaaa?gaaacactaa 1860

gctctccggg?cctggcttac?tggggaagcc?acgcagggcc?tgggctgagt?gccgagcagc 1920

cctagccaca?gggtccctgg?gggaggccgc?ccacccgagg?ctgaggctgg?caccccatgg 1980

ctgagcggac?cccgctcctg?cagcatgcct?cagccccaca?gacgcccacc?cctcttttgt 2040

ttttctttgc?accagtcttc?caggccagtg?ccactgtgct?ggctgctggc?ggatgccccc 2100

agcctggatg?gaggtgggat?tccctccagg?tgggggcgcc?cacaccccca?ttgaagagga 2160

gcatgcctca?agggagcagt?tggtagggaa?ggcagtgggc?agcagacttg?attctgaccc 2220

caggccttgg?gtgggtcctc?cctcagcacc?ccactctctc?cagcctctgc?agcagccact 2280

gagccctgcc?cacgctgtgt?cagcatcgca?cctcccacct?ccgcagcacc?ccggcttggc 2340

ctcagccacg?cccttctttc?tccagccggg?cgacactggc?tccagcccag?ctgaagcgca 2400

cactccctgg?agcgcctcca?gcacacgcag?cacgagcact?gaaatcactt?tacctgcagg 2460

ttccacaacc?tcggcctccc?tcctgaggca?ggtggaccac?agagctgtgc?ccctgactcc 2520

ccgggcgggc?ggggagccct?gctgccccag?cccagcactg?ctcgcagggg?aggtacccag 2580

gatgaactga?tcccgctcac?ttgtgacacc?catttgttcc?agcagctctg?ctgccctccc 2640

ctttccttgt?gattggccca?gccaggcacc?tggagctctc?cctgcaccgc?ttctggtgac 2700

cagggaccct?gccaggc 2717；

(2) amino acid of SEQ ID No:1 in the code sequence tabulation, it is made up of 441 amino-acid residues, and its sequence is as follows:

MEQPPEEAPE?VREEEKKKEV?AEAEGGPELN?GEPEHSLPSS?SCTDLSQSCS?PPALLDQLQM 60

GCDGASCGGL?SMECRVCGDK?ASGFHYGVHA?CEGCKGFFRR?TIRMKLEYEK?CERICKIQKK 120

NRNKCQYCRF?QKCLALGMSH?NAIRFGRMPE?AEKRKLVAGL?TANEGSQHNP?QVADLKAFSK 180

HLYSAYLKNF?NMTKKKARAI?LTGKASHTAP?FVIHDIETLW?QAEKGLVWKQ?LVNGLPPYKE 240

ISVHVFYRCQ?CTTVETVREL?TEFAKSIPSF?DHFFLNDQVT?LLKYGVHEAI?FAMLASIVNK 300

DGLLVANGTG?FVTREFLRSI?RKPFSDIIEP?KFEFAVKFNA?LELDDSDLAL?FIAAIILCGD 360

RPGLMNVSQV?EAIQDTILRA?LEFHLQANHP?DAQYLFPKLL?QKMADLRQLV?TEHAQMMQRI 420

KKTETETSLH?PLLQEIYKDM?Y 441；

(3) with sequence table in the nucleotide sequence that limits of SEQ ID No:2 have 90% above homology, and the proteinic nucleotide sequence of identical function of encoding;

(4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID No:2 in the sequence table.

2. major gene as claimed in claim 1 is characterized in that: the encoding sequence of described cDNA sequence is the base from the 102nd to 1427 at 5 ' end of SEQ ID No:2 in the sequence table.

3. major gene as claimed in claim 1, it is characterized in that: the proteinic name that major gene is translated is called peroxisome proliferation-activated receptors D, derive from pig, amino acid residue sequence with SEQ ID No:1 in the sequence table, through replacement, disappearance or the interpolation of 1-10 amino-acid residue and with pig remebrance shape, fatty deposits, nose heave and trunk grow proterties relevant by SEQ ID No:1 deutero-protein.

4. molecular breeding method at boar remebrance shape, fatty deposits, the nose heave and long proterties of trunk is characterized in that:

(1), the nucleotide sequence of SEQ ID No:3 and SEQ ID No:4 in the use sequence table, pig genomic dna to be measured is carried out pcr amplification, dyeing is carried out single nucleotide polymorphism to pcr amplification product and is detected, and determines that from 5 ' end the 565th bit base of SEQ ID No:3 or 5 ' end the 121st bit base of SEQ ID No:4 be G or A;

(2) if the mononucleotide polymorphic result of pcr amplification product holds the 121st bit base (promptly holding the 565th bit base from 5 ' of SEQ ID No:3) to be G from 5 ' of SEQ ID No:4, its homozygotic genotype is GG; 5 ' end the 121st bit base (promptly holding the 565th bit base from 5 ' of SEQ ID No:3) from SEQ ID No:4 is A, and its homozygotic genotype is AA; Their heterozygote genotype is GA; The individual ear area of AA genotype, ear weigh, trunk is long and the nose heave utmost point is higher than GA genotype and GG genotype individuality significantly, significantly are being lower than GA genotype and GG genotype individuality aspect the fatty deposits proterties (the average thickness of backfat).

5. the application of major gene in kind of swine improvement of pig remebrance shape, fatty deposits, the nose heave and long proterties of trunk.

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