CN103898107A - Major SNP (single nucleotide polymorphism) marker influencing growth traits of pigs and application thereof in genetic improvement of productivity of breeding pigs - Google Patents
Major SNP (single nucleotide polymorphism) marker influencing growth traits of pigs and application thereof in genetic improvement of productivity of breeding pigs Download PDFInfo
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Abstract
The invention provides a major SNP (single nucleotide polymorphism) marker influencing the growth traits of pigs. The SNP marker is located at a nucleotide sequence of an HMGA1 gene of a pig chromosome 7, a locus of the SNP marker is the nucleotide mutation of C857-G857 with an SEQ ID NO:1 sequence labeling position of 857, corresponding to a 34983991st nucleotide locus C>on chromosome 7 in a reference sequence in an international pig genome version 10.2, G mutation, or one of seven other loci completely linked with the locus. The invention also provides the application of the SNP marker in the genetic improvement of growth traits of breeding pigs, and provides the application of an SNP molecular marker with a linkage disequilibrium degree (r2) with the SNP marker of greater than 0.8 in the genetic improvement of growth traits of breeding pigs. The growth traits include one or more of the length and height of a living body of a pig, carcass length, carcass weight, daily gain and head weight. According to the invention, the breeding process of breeding pigs can be accelerated, the productivity of breeding pigs can be effectively improved, and remarkable economic benefits can be obtained.
Description
Technical field
The present invention relates to animal molecular biotechnology and Animal Genetics field, to the effect that differentiate and can significantly increase pig live body body length, live body height, trunk length, carcass weight, day weight gain, the major gene mark of the growth traits such as nose heave and the application in boar production performance genetic improvement thereof.
Background technology
Growth traits is to evaluate the important indicator of pig production performance, is therefore the free-revving engine proterties in kind of swine improvement research, and it mainly comprises trunk length, carcass weight, day weight gain, nose heave, the thickness of backfat, eye muscle area etc.Traditional pig breeding method is to assess by the compatriot to object individuality or descendant's slaughter determining, and accuracy is higher, achieves noticeable achievement, but exist, cost of determination is high, the deficiency of much time power.Genetic improvement method economy based on major gene molecule marker, quick and effectively, be the necessary complement to traditional pig breeding.
Up to now, the molecule marker that affects pig growth traits and the major gene of existing some amount are identified, have greatly promoted the progress of international seed swine improvement.(the Science.1991Jul26 such as Fujii; 253 (5018): 448-51) find that Hal gene is to cause pig to produce the major gene of greyish white water sample meat (Pale, soft, exudative pork, PSE meat); (the Anim Genet.1997Apr such as Casas-Carrillo; 28 (2): 88-93) finding to be positioned at IGF-1 gene and daily postweaning gain on No. 5 karyomit(e)s of pig exists chain; Milan(Science.2000May19; 288 (5469): 1248-51) etc. identified that PRKG3 is the major cause that causes sour meat in hampshire colony; Van Laere(Nature.2003Oct23; 425 (6960): 832-6) thus etc. research find that the growth of IGF2 regulation and control pig muscle affects the meat yield of business population 10～20%.The rapid application on producing of the discovery of these major genes and correlation detection technology, plays important pushing effect to the development of international boar industry.
China is one of country of raising pigs in the world the earliest, and history is for several thousand, and pig is described as first of six domestic animals in China.Due to the impact of physical environment, economic condition and folk culture, China local pig breed aboundresources and differ from one another, be the precious resources in world's pig kind gene pool.But up to now, not yet in Chinese native pig breed, identify major gene and the molecule marker relevant to growth traits.
Applicant in earlier stage utilize constructed extensive white duroc × Erhualian F2 resource colony, measured 927 F2 and butchered the long phenotype of individual trunk.The major gene loci (QTL) that affects hog on hook length on No. 7 karyomit(e)s detected by full genome scanning, the interval size of Primary Location is 3cM(centimorgan), this is for later separation and differentiate that affecting trunk long major gene and molecule marker provides important point of penetration.
Summary of the invention
The invention provides a kind of main effect SNP mark that affects pig growth traits, the nucleotides sequence that described SNP mark is positioned at High mobility group A1 (HMGA1) gene on No. 7 karyomit(e)s of pig lists, the site of described SNP mark is the coding mutation of the SEQ ID NO:1 sequence labelling position C857-G857 that is 857, corresponding to the 34983991st nucleotide site C>G sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position T3651-G3651 that is 3651, corresponding to the 34986785th nucleotide site T>G sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C3676-T3676 that is 3676, corresponding to the 34986810th nucleotide site C>T sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C3737-T3737 that is 3737, corresponding to the 34986871st nucleotide site C>T sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position G4803-A4803 that is 4803, corresponding to the 34987937th nucleotide site G>A sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position G5652-C5652 that is 5652, corresponding to the 34988786th nucleotide site G>C sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C5784-A5784 that is 5784, corresponding to the 34988918th nucleotide site C>A sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position TT6965_6966-TTdel6965_6966 that is 6965-6966, corresponding to 34990100-34990101 nucleotide site TT deletion mutantion on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Aforementioned 8 site complete linkages.
The present invention also provides a kind of SNP as above to be marked at the application in boar growth traits genetic improvement, and described growth traits comprises pig live body body length, live body height, trunk length, carcass weight, day weight gain, one or more in nose heave.According to described application, described boar from place of china pig and the synthetic system of containing place of china pig blood lineage and supporting be pig kind.Be preferably selected from Erhualian, Dongshan Swine, people pig, the large ear pig of the river bend, pig is hidden in Tongcheng pig, Rongchang Pig, Neijiang Pig, sand ridge pig, laughable pig, Guangdong Arabescato pig, Luchuan pig, the black pig in Gan Xi two, enlightening celebrating Tibetan pig, the fragrant pig of bar horse, Mingguang City microtia pig, eight eyebrow pigs, Tibetan, Milin pig, Wuzhi Mountain pig, the southern regions of the Yunnan Province microtia pig, the fragrant pig in the Congjiang, Tibetan, Litang pig, cooperation, pig, Laiwu Pigs and Su Tai pig are hidden in Gongbo'gyamda.
The present invention also provides the linkage disequilibrium degree (r of a kind of and above-mentioned SNP mark
2) be greater than the application in boar growth traits genetic improvement of 0.8 SNP molecule marker.
The present invention provides again the method for a kind of kind of swine improvement, it is characterized in that the GG type individuality in site g.34983991 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of boar subculture seed selection is eliminated the individual and CG type individuality of the CC type in this site; And/or the GG type individuality in site g.34986785 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and TG type individuality of the TT type in this site; And/or the TT type individuality in site g.34986810 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CT type individuality of the CC type in this site; And/or the TT type individuality in site g.34986871 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CT type individuality of the CC type in this site; And/or the AA type individuality in site g.34987937 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and AG type individuality of the GG type in this site; And/or the CC type individuality in site g.34988786 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CG type individuality of the GG type in this site; And/or the AA type individuality in site g.34988918 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and AC type individuality of the CC type in this site; And/or the del/del type individuality in site g.34990100-34990101 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and TT/TT type individuality of the TT/del type in this site.
The present invention also provides a kind of method of the production performance that detects pig, the 857th Nucleotide of 5 ' end that comprises SEQ ID NO:1 in detection sequence table is C or G, or the 3651st Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is T or G, or the 3676th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or T, or the 3737th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or T, or the 4803rd Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is G or A, or the 5652nd Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is G or C, or the 5784th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or A, or detecting 5 ' the end 6965-6966 position Nucleotide of SEQ ID NO:1 in sequence table is TT or del, determine genotype, then determine the production traits by genotype, and the production traits of described pig is to comprise pig live body body length, live body height, trunk length, carcass weight, day weight gain, one or more the growth traits of pig in nose heave.
The invention provides a kind of nucleotide sequence that is positioned at the HMGA1 gene on No. 7 karyomit(e)s of pig, described sequence be SEQ ID NO:1 the 857th Nucleotide by C sport G, 3651 Nucleotide by T sport G, 3676 Nucleotide by C sport T, 3737 Nucleotide by C sport T, 4803 Nucleotide by G sport A, 5652 Nucleotide by G sport C, 5784 Nucleotide by C sport A, 6965-6966 position Nucleotide TT lacks.
The present invention also provides a kind of described nucleotides sequence to be listed in and application in pig live body body length, live body height, trunk length, carcass weight, day weight gain and/or nose heave relevant boar seed selection, the synthetic system that preferred described boar is selected from place of china pig and contains place of china pig blood lineage and supporting be pig kind.
Brief description of the drawings
Fig. 1 affects the Fine Mapping figure of the long site of trunk in white duroc × painted face in Beijing opera F2 resource colony on No. 7 karyomit(e)s of pig;
Fig. 2 is the g.34986810C>T sequencing result peak figure of different genotype of HMGA1 gene main effect mutational site.
Embodiment
Below in conjunction with embodiment and contrast accompanying drawing the present invention is described in further detail.
Experiment swinery: the present invention has used two pig experimental populations altogether: white duroc × Erhualian F
2the purebred colony of resource colony and Erhualian.White duroc × Erhualian F
2f breeds for generations taking 2 white Duroc boars and 17 Erhualian sows as F0 in resource colony
1generation, then select 9 F
1for boar and 59 F
1sow mating, point 1912 F of 6 batches of generations
2in generation, is individual, wherein 1031 individual feeding to 240 ± 3 age in days slaughter determinings.Erhualian colony is purchased from burnt small stream painted face in Beijing opera conservation field, changzhou, and wherein 336 individualities are at 300 ± 3 age in days slaughter determinings.Above-mentioned all experiment pigs are all raised on Agricultural University Of Jiangxi's Animal Biotechnology key lab scientific research pig farm, and 10～12 pigs of grow-finish stage every hurdle stable breeding, (every pig area occupied is 2m
2), free choice feeding drinking-water, and by unified feeding standard.Every kilogram of diets of different nutrition level is: crude protein 16%, digestible energy 13.1MJ, Methionin 0.78%, calcium 0.6%, phosphorus 0.5%.
Phenotype test: phenotype test carries out in Jiangxi green food source mill of Guo Hong Group Co.,Ltd, pig is jejunitas first 20 hours of government official, but can freely drink water, whole slaughtering process implements according to normal business program.Before killing, measure individual live body body weight, live body body length and live body height; Live body body length is the length of mid point along lineback to root of the tail that measures live hog two basal part of the ear on lines with tape measure; Live body height is to measure the vertical range on pig hair worn in a bun or coil first place to ground with biltmore stick; Pig butcher rear along basal part of the ear trailing edge and lower jaw Article 1 nature tuck cut off extensive region, pillow joint separates pig's head, measures nose heave; Carcass weight is the weight of the two trunk sums in left and right (comprising leaf fat and kidney); Trunk directly length is the length from pubic symphysis leading edge mid point to atlas leading edge mid point; Trunk plagioclase is the length from pubic symphysis leading edge mid point to the first rib and breastbone junction; Day weight gain is killed the difference of front live body body weight and birth weight by calculating, then obtains divided by slaughter age.
Embodiment 1
The present embodiment is the invention process that specific explanations obtains genetic marker in the present invention.
The full genome 60K of pig SNP genotype detection: the each individual fritter ear tissue sample that gathers from above-mentioned two experimental populations, extract genomic dna with standard phenol chloroform method, DNA is dissolved in TE damping fluid.With Nanodrop-1000 nucleic acid-protein analyser to white duroc × Erhualian F
2the DNA of resource colony carries out quality examination and concentration determination, and A260/280 ratio is 1.8-2.0, and it is qualified that A260/230 ratio is judged to be 1.7-1.9.Qualified DNA sample unification is diluted to 50ng/ μ L
-1utilize Illumina Infinium SNP somatotype platform, order Porcine SNP60DNA Analysis Kit chip, carry out chip hybridization and result scanning according to the operation instruction of Illumina Infinium and normal process, read genotype data by GenomeStudio software.With PLINK v1.07 to obtain genotype data carry out quality control, reject recall rate <99.7%, inferior gene frequency (minor allele frequency, MAF) <0.01 or depart from Hardy Weinberg balance (Hardy – Weinberg Equilibrium, HWE) P≤10
-6sNP mark, get rid of recall rate <90%, family Mendelian's error rate higher than 0.1 individuality, finally have 48165 SNP and 1018 individualities for data analysis.
Full genome association (GWAS) is analyzed, the determining of Fine Mapping and candidate gene: use the mixed linear model in R software package GenABEL, utilize white duroc × Erhualian F
260K SNP marker genetype data and the trunk phenotypic data of all individualities of resource colony carry out GWAS analysis, and model is: y=1 μ+Xb+Sc+Z α+e.Wherein, y is measured phenotypic number, and μ is population mean, b be sex and batch fixed effect vector, c answers c-N(0, σ for remaining minor effect
c 2), α is random additive genetic effect vector, and α～N(0, G σ
α 2) (G is the molecule blood relationship correlation matrix between individuality, σ
α 2for additive genetic variance), e is residual error, X, S and Z are the incidence matrix to fixed effect and random benefit, year season (herd-year-season) effect be included in batch effect.Adopt Bonferroni method to determine the significance threshold value of SNP and growth traits correlation degree, genomic level remarkable threshold is 0.05 divided by effective SNP site quantity, i.e. 0.05/48165=1.04e-6; Chromosome level remarkable threshold is 1 divided by effective SNP site quantity, i.e. 1/48165=2.08e-5.GWAS result shows, has the SNP site (Figure 1A) with the long significant correlation of trunk on No. 7 karyomit(e)s of pig.Adopt chain and linkage disequilibrium analysis (LDLA) method integrated, by the most remarkable site-log
10(P) value decline 2 fiducial intervals that define QTL are 1.3Mb(Figure 1B).
The auxiliary method for separating and analyzing of applying marking is judged 9 F1 boar QTL genotype of F2 resource colony.Its principle is: the QTL genotype of every F1 boar is by the decision of Z value, and Z value is LH
1/ LH
0the Log of likelihood ratio ratio
10value; Wherein LH
1to suppose descendant's phenotype DATA DISTRIBUTION probability when boar is Qq heterozygote, LH
0to suppose descendant's phenotype DATA DISTRIBUTION probability when boar is QQ or qq homozygote.In the time of Z<-2, QTL genotype is QQ or qq; In the time of Z>2, QTL genotype is Qq; In the time of-2<Z<2, QTL genotype be can not determine.Calculation result shows that 8 F1 boars are Qq heterozygote (table 1), and Q all derives from painted face in Beijing opera, i.e. E
q.The QTL genotype of 1 F1 boar be can not determine.Table 1 has provided the result table that white Du Luoke × painted face in Beijing opera F1 boar QTL genotype is judged.
Table 1
In table 1,
1for containing the chromosomal F2 offspring number of individuals in F1 boar left side;
2for containing F1 boar left side karyomit(e) F2 offspring's phenotype mean value;
3for containing F1 boar right side chromosomal F2 offspring number of individuals;
4for containing F1 boar right side karyomit(e) F2 offspring's phenotype mean value;
5for all F2 offspring number of individuals of F1 boar.
Utilize the SNP haplotype of No. 7 karyomit(e) objects of DualPHASE software building and neighboring area 34.5-36.5Mb scope, by judging the shared haplotype of painted face in Beijing opera Q karyomit(e), QTL is positioned between SNP ss131343640 and SNPss478941605.In painted face in Beijing opera haplotype analysis, find that wherein 4 individualities have carried and E for generations at 17 F0
qtwo kinds of different haplotypes, judge that by haplotype effect analysis the QTL genotype of these two kinds of haplotypes is q, i.e. E
q.Get rid of E
qwith E
qshared region, can be contracted to QTL region the scope (Fig. 1 C) of about 475kb between SNP ss131343774 and SNP ss131344094.
Equally, utilize Chinese native pig breed 60K data, build the haplotype between SNP ss131343774 and SNPss131344094 at No. 7 karyomit(e)s.Found that and in most of Chinese native pig breed, all contain E
qhaplotype, wherein Dongshan Swine, people pig, the large ear pig of the river bend, the E of the pig kinds such as Tongcheng pig, Rongchang Pig, Neijiang Pig, sand ridge pig, laughable pig, Guangdong Arabescato
qhaplotype frequency is far above 50%(table 2).This illustrates E
qin Chinese native pig breed, have obvious separation, therefore the major gene in this site and molecule marker have important seed selection using value in numerous place of china pigs and place of china pig blood lineage's synthetic system and supporting system.Table 2 is the E of the each pig kind of place of china
qhaplotype distribution frequency, wherein " quantity " refers to a number of this pig kind of research, and " frequency " refers to E
qthe percentage composition of haplotype in total monoploid (respective numbers 2 times).
Table 2
The meticulous region of this QTL is corresponding to the 34776047bp(ss131343774 on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version)) to 35251345bp(ss131344094) interval.This 475kb interval is contained 5 and has been annotated gene, is respectively: GRM4, HMGA1, RPS10, SPDEF and PACSIN1.Utilize bioinformation method to carry out gene function analysis to above 5 candidate genes, wherein the albumen of HMGA1 coded by said gene is a member in high migration protein family, (the Dev Cell.2002 such as Karsenty; 2 (4): 389-406) and Richter etc. (Tissue Eng Part A.2009; 15:473-7.) research shows that HMGA1 albumen is conducive to improve chondrocyte proliferation activity at external its of pig cartilage, is one of important step of bone length growth.Wu etc. (J Biol Chem.2004279:37485-90.) find this gene specifically expressing in the appendicular skeleton of animal and scleroblast, think that this gene may play critical effect in the growth course of bone cells.(the Nat Genet.2008 such as Gudbjartsson; 40:609-15.) the intragenic sudden change of HMGA1 and the mankind's height significant correlation is found in research.In addition (the Mol Endocrinol.2012 such as Iiritano; 26:1578-89.) disclosing HMGA1 albumen can be by regulating IGF1 biological activity to carry out the picked-up of affecting glucose.Above-mentioned data shows, HMGA1 gene plays an important role in the growth to bone and carbohydrate metabolism in animal growth process, be this region most possibly affect the proterties such as trunk is long because of fruit gene (causative gene).
The target gene order of resurveying: the about 5.4Kb of HMGA1 mrna length, has 4 exons.In Ensembl website, (http://asia.ensembl.org/index.html) downloads the genomic dna sequence of HMGA1, utilizes the online design software Primer3.0(http of primer: //frodo.wi.mit.edu/) 16 pairs of primers of design (in table 3).With white duroc × Erhualian F
2in resource colony, determine that QTL genotype comprises painted face in Beijing opera Q(E for generations
q), painted face in Beijing opera q(E
q) and white Du Luoke q(D
q), choose genotype E
qe
qindividual 1, E
qe
qindividual 2, E
qd
qindividual 1 and D
qd
qindividual 2 totally 6 individualities be template, the section of amplification HMGA1 genetic transcription initiation site upstream 2kb and Transcription Termination site downstream 2kb left and right.In polymerase chain reaction (PCR) reaction system of 50ul, comprise the pig genomic dna of 100ng, 2.5mM MgCl
2, 0.4mM dNTP, the each 20pmol of forward and reverse primer, 2.5 unit archaeal dna polymerases (Taq enzyme) and 10*PCR buffer(damping fluid) and (Shanghai Shen Neng lottery industry company limited).PCR adopts Touchdown program, and amplification condition is: 94 DEG C of 5min; 94 DEG C of 30s, 68 DEG C of (0.5 DEG C of each cycle down) 30s, 72 DEG C of 60s.94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, 18 circulations; Finally extend 10min at 72 DEG C.Pcr amplification product is delivered to 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.25 polymorphic sites detected altogether, comprising 24 SNP and a disappearance insertion point.
Table 3
The genotypic consistency analysis of HMGA1 gene polymorphic loci gene type and QTL: above-mentioned 6 F of comparative analysis
225 polymorphic site genotype that the individual HMGA1 gene of resource family resurveys that order obtains and QTL genotype, get rid of the SNP not being inconsistent with QTL genotype, result detects 8 sites (table 4) consistent with QTL genotype altogether: g.34983991C>G, g.34986785T>G, g.34986810C>T, g.34986871C>T, g.34987937G>A, g.34988786G>C, g.34988918C>A, g.34990100_34990101TTdel.Wherein, etc. g.34983991C>G the 34983991st nucleotide site, i.e. the 857th Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34986785T>G the 34986785th nucleotide site, i.e. the 3651st Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34986810C>T the 34986810th nucleotide site, i.e. the 3676th Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34986871C>T the 34986871st nucleotide site, i.e. the 3737th Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34987937G>A the 34987937th nucleotide site, i.e. the 4803rd Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34988786G>C the 34988786th nucleotide site, i.e. the 5652nd Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34988918C>A the 34988918th nucleotide site, i.e. the 5784th Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in mutational site; G.34990100_34990101TT 34990100-34990101 nucleotide site, i.e. 6965-6966 Nucleotide of following SEQ ID NO:1 sequence on No. 7 karyomit(e)s of international pig genome reference sequences (10.2 version) that announce corresponding to Ensembl website in disappearance (del) mutational site.Table 4 has been listed affects hog on hook long HMGA1 gene polymorphic site and the genotypic consistency analysis of QTL, wherein 1～8 represent respectively above-mentioned 8 mutational sites, " del/del " in table 4 represents that mutational site genotype is g.34990100_34990101 " TT disappearance/TT disappearance ", " del/-" represents that mutational site genotype is g.34990100_34990101 " TT disappearance/TT ", "/-" represent that mutational site genotype is g.34990100_34990101 " TT/TT ".
Table 4
The affect effect analysis of main effect mutational site on growth traits: due to above-mentioned 8 mutational site complete linkages, so correlation analysis is carried out with hog on hook phenotype in 1 the main effect mutational site the present invention is directed to wherein g.34986810C>T.After employing Auele Specific Primer pcr amplification, the method for direct Sequencing detects the g.34986810C>T genotype in mutational site.Upstream and downstream amplimer is respectively 5 '-CGA AGT GCC AAC ACC TAA AAG-3 ' and 5 '-TGG AGC TGT GGT GGT TTT C-3 '.In polymerase chain reaction (PCR) reaction system of 50ul, comprise the pig genomic dna of 100ng, 2.5mM MgCl
2, 0.4mM dNTP, the each 20pmol of forward and reverse primer, 2.5 unit archaeal dna polymerases (Taq enzyme) and 10 × PCR buffer(damping fluid) and (Shanghai Shen Neng lottery industry company limited).PCR adopts Touchdown program, and amplification condition is: 94 DEG C of 5min; 94 DEG C of 30s, 68 DEG C of (0.5 DEG C of each cycle down) 30s, 72 DEG C of 60s; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, 18 circulations; Finally extend 10min at 72 DEG C.Pcr amplification product is delivered to 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.Sequencing result as shown in Figure 2, different genotype on the corresponding arrow of the peak figure of different colours.G.34986810C>T site be blue unimodal this individual be CC type homozygote, if occur blue and red bimodal be CT type heterozygote, if only occur redness unimodal be TT type homozygote; As shown in table 5, at white duroc × Erhualian F
2in resource colony, g.34986810C>T there is utmost point significant correlation in site and multiple growth traits, wherein TT genotype is individual compares with CC genotype individuality: the longer 9.28cm of the 210 long mean value of age in days live body body, the higher 4.86cm of 210 age in days live body height mean value, 240 age in days trunks are the longer 9.08cm of long mean value directly, the longer 7.26cm of 240 age in days trunk plagioclase mean value, the heavier 7.67kg of 240 age in days carcass weight mean value, the heavier 33.84g of 0 to 240 age in days day weight gain mean value, the heavier 2.03kg of the nose heave mean value of 240 age in days.Other 7 polymorphic sites and g.34986810C>T site complete linkage, therefore on growth traits to affect effect identical.Table 5 has been listed the effect that affects on 240 age in days pig growth traitss in white duroc × Erhualian F2 resource colony of mutational site g.34986810C>T.
Table 5
The present embodiment is the g.34986810C>T application in boar seed selection of SNP site obtaining in embodiment 1.According to E
qhaplotype is in the distribution frequency analysis of Chinese native pig breed, for the nucleus herds of breeding pigs individuality of place of china pig and the synthetic system that comprises place of china pig blood relationship and supporting system, utilize the method for direct Sequencing after above-mentioned pcr amplification to detect the g.34986810C>T genotype of main effect mutational site that affects pig growth traits on No. 7 karyomit(e)s of pig, select beneficial gene type TT to reserve seed for planting to individuality, after colony's subculture seed selection, increase live body body length, live body height, trunk length, carcass weight, day weight gain and the growth performance such as nose heave of swinery.
In the present embodiment, utilize painted face in Beijing opera colony for laboratory animal.334 Erhualian genomic dnas are carried out to direct Sequencing after pcr amplification with Auele Specific Primer.According to sequencing result, main effect marker site genotype is g.34986810C>T judged.Then utilize R software to carry out the effect analysis that affects of genotype on phenotype, model is: y=Z α+e, and y is measured phenotypic number, α is random additive genetic effect vector, and α～N(0, G σ
α 2) (G is molecule blood relationship correlation matrix between individuality, σ
α 2for additive genetic variance), e is residual error, Z is the incidence matrix of random benefit.Table 6 is that g.34986810C>T mutational site affects effect table to 300 age in days growth traitss in Erhualian.As seen from Table 6, g.34986810C>T TT genotype in site is individual compares with CC genotype individuality: the longer 12.67cm of the 300 long mean value of age in days live body body, the higher 5.7cm of 300 age in days live body height mean value, 300 age in days trunks are the longer 9.94cm of long mean value directly, the longer 6.87cm of 300 age in days trunk plagioclase mean value, the heavier 13.09kg of 300 age in days carcass weight mean value, the heavier 61.11g of 0 to 300 age in days day weight gain mean value, the heavier 1.91kg of the nose heave mean value of 300 age in days.Other 7 polymorphic sites and g.34986810C>T site complete linkage, therefore on growth traits to affect effect identical.As can be seen here, in painted face in Beijing opera colony and the synthetic system of containing painted face in Beijing opera blood relationship, supporting system (table 6), seed selection is the TT type individuality in site g.34986810C>T, can improve significantly live body body length, live body height, trunk length, carcass weight, the day weight gain and nose heave of swinery by the utmost point, improve growth performance.
Table 6
Claims (10)
1. affect the main effect SNP mark of pig growth traits, it is characterized in that, the nucleotides sequence that described SNP mark is positioned at High mobility group A1 (HMGA1) gene on No. 7 karyomit(e)s of pig lists, the site of described SNP mark is the coding mutation of the SEQ ID NO:1 sequence labelling position C857-G857 that is 857, corresponding to the 34983991st nucleotide site C>G sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position T3651-G3651 that is 3651, corresponding to the 34986785th nucleotide site T>G sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C3676-T3676 that is 3676, corresponding to the 34986810th nucleotide site C>T sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C3737-T3737 that is 3737, corresponding to the 34986871st nucleotide site C>T sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position G4803-A4803 that is 4803, corresponding to the 34987937th nucleotide site G>A sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position G5652-C5652 that is 5652, corresponding to the 34988786th nucleotide site G>C sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position C5784-A5784 that is 5784, corresponding to the 34988918th nucleotide site C>A sudden change on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Or be the coding mutation of the SEQ ID NO:1 sequence labelling position TT6965_6966-TTdel6965_6966 that is 6965-6966, corresponding to 34990100-34990101 nucleotide site TT deletion mutantion on No. 7 karyomit(e)s of international pig genome 10.2 version reference sequences; Aforementioned 8 site complete linkages.
2. SNP as claimed in claim 1 is marked at the application in boar growth traits genetic improvement, and described growth traits comprises pig live body body length, live body height, trunk length, carcass weight, day weight gain, one or more in nose heave.
3. application according to claim 2, is characterized in that, the synthetic system that described boar is selected from place of china pig and contains place of china pig blood lineage and supporting be pig kind.
4. application according to claim 3, it is characterized in that, described boar is selected from Erhualian, Dongshan Swine, people pig, the large ear pig of the river bend, and pig is hidden in Tongcheng pig, Rongchang Pig, Neijiang Pig, sand ridge pig, laughable pig, Guangdong Arabescato pig, Luchuan pig, the black pig in Gan Xi two, enlightening celebrating Tibetan pig, the fragrant pig of bar horse, Mingguang City microtia pig, eight eyebrow pigs, Tibetan, Milin pig, Wuzhi Mountain pig, the southern regions of the Yunnan Province microtia pig, the fragrant pig in the Congjiang, Tibetan, Litang pig, cooperation, pig, Laiwu Pigs and Su Tai pig are hidden in Gongbo'gyamda.
With the HMGA1 gene of claim 1 in the linkage disequilibrium degree (r of SNP mark
2) be greater than the application in boar growth traits genetic improvement of 0.8 SNP molecule marker.
6. a method for kind swine improvement, is characterized in that, the GG type individuality in site g.34983991 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of boar subculture seed selection is eliminated the individual and CG type individuality of the CC type in this site; And/or the GG type individuality in site g.34986785 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and TG type individuality of the TT type in this site; And/or the TT type individuality in site g.34986810 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CT type individuality of the CC type in this site; And/or the TT type individuality in site g.34986871 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CT type individuality of the CC type in this site; And/or the AA type individuality in site g.34987937 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and AG type individuality of the GG type in this site; And/or the CC type individuality in site g.34988786 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and CG type individuality of the GG type in this site; And/or the AA type individuality in site g.34988918 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and AC type individuality of the CC type in this site; And/or the del/del type individuality in site g.34990100-34990101 on No. 7 karyomit(e)s of the international pig genome 10.2 version reference sequences pigs of subculture seed selection, eliminate the individual and TT/TT type individuality of the TT/del type in this site.
7. one kind is detected the method for the production performance of pig, the 857th Nucleotide of 5 ' end that comprises SEQ ID NO:1 in detection sequence table is C or G, or the 3651st Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is T or G, or the 3676th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or T, or the 3737th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or T, or the 4803rd Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is G or A, or the 5652nd Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is G or C, or the 5784th Nucleotide of 5 ' end that detects SEQ ID NO:1 in sequence table is C or A, or detecting 5 ' the end 6965-6966 position Nucleotide of SEQ ID NO:1 in sequence table is TT or del, determine genotype, then determine the production traits by genotype, and the production traits of described pig is to comprise pig live body body length, live body height, trunk length, carcass weight, day weight gain, one or more the growth traits of pig in nose heave.
8. be positioned at a nucleotide sequence for the HMGA1 gene on No. 7 karyomit(e)s of pig, described sequence be SEQ ID NO:1 the 857th Nucleotide by C sport G, 3651 Nucleotide by T sport G, 3676 Nucleotide by C sport T, 3737 Nucleotide by C sport T, 4803 Nucleotide by G sport A, 5652 Nucleotide by G sport C, 5784 Nucleotide by C sport A, 6965-6966 position Nucleotide TT lacks.
9. a nucleotides sequence as claimed in claim 8 is listed in and application in pig live body body length, live body height, trunk length, carcass weight, day weight gain and/or nose heave relevant boar seed selection.
10. application according to claim 9, is characterized in that, the synthetic system that described boar is selected from place of china pig and contains place of china pig blood lineage and supporting be pig kind.
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