CN103320516A - Method and special product for assisted identification of swine backfat thickness character - Google Patents
Method and special product for assisted identification of swine backfat thickness character Download PDFInfo
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Abstract
The invention discloses a method and a special product for the assisted identification of swine backfat thickness character. The method for the assisted identification of swine backfat thickness character comprises the following steps of: determining whether the deoxyribonucleotide 47033 of the GenBank Accession Number GU565976.1 of a peroxisome proliferator-activated receptor delta gene of a to be detected swine is T, or C, or T and C, so as to determine whether the genotype of the to be detected swine is TT or TC or CC, and determining the backfat thickness character according to the genotype of the to be detected swine, wherein the backfat thickness of the to-be-detected swine with the TT genotype is greater than that of the to be detected swine with the TC genotype, and the backfat thickness of the to be detected swine with the TC genotype is greater than that of the to be detected swine with the CC genotype. The method and the special product disclosed by the invention are used for breeding swine, so that the to be detected swine can be screened in the early stage, the problem of long time needed for selecting good breeding swine in the practical production can be effectively solved, the breeding cost is reduced, and the swine backfat thickness in the practical production is effectively reduced or increased.
Description
Technical field
The present invention relates to a kind of method and special product thereof of assistant identification fat thickness at back of pig proterties.
Background technology
The big good characteristic that China's local pig breed is well-known is exactly that meat is of fine quality good, commodity pork is hybridized all because of its good meat matter in these local variety, the cultivation kind that contains local variety blood or even China and foreign countries, extensively get consumer reception, for example black pork, Black Hills pork of Beijing Black pork, Laiwu, the pork of reviving too etc. are subjected to human consumer's favorable comment deeply.And the price of these high-quality porks is 1.5-3 times of common pork, has demonstrated fully the commercial value of current generation China's local variety pig.But these pig kind majorities all are lard type, and its distinguishing feature is that back-fat thickness is big.The back fat deposition too much not only causes poor growth, does not also meet existing situation simultaneously for the requirement of joint grain.As everyone knows, growth lean meat is compared the fatty required little energy of growth, and the most local variety pigs of China are lard type, and its feedstuff-meat ratio majority is all more than 4:1.Show according to the study, the genetic correlation of the thickness of backfat and feedstuff-meat ratio can reach 0.55(Hoque MA, Suzuki1K, Kadowaki H, Shibata T, Oikawa T.Genetic parameters for feed efficiency traits and their relationships with growth and carcass traits in Duroc pigs.J Anim Breed Genet.2007,124:108-116.), can reach-0.58(Sun Hua with the genetic correlation of lean ratio, Song Zhongxu, Li Lianghua, Peng Xianwen, Guo Wanzheng, military Hua Yu, Mei Shuqi. Chinese Large White S II 1 owner will grow and the genetic parameter of carcass trait is estimated. hubei agricultural science .2009,48,3086-3089.).Therefore, China's local pig breed and the kind thickness of backfat that contains local pig blood lineage are all more than 3 centimetres, and the advantage of China's local variety is not also given full play of, and compare with international business pig kind, and potentiality also are significantly improved.
Based on the importance of above-mentioned fat thickness at back of pig proterties, carry out the emphasis that breeding work is current breeding work at it.Because traditional breeding method speed is slow, order of accuarcy is low, molecular breeding has become current breeding work major technique means with characteristics such as its accuracy and rapidities.
Peroxisome proliferation-activated receptors (PPARs) is the nuclear factor that a class is activated by part, belong to steroid/Tiroidina/retinoid receptor superfamily, by PPAR α, PPAR γ and 3 member compositions of PPAR δ (PPARD), they can both mediate lipotropy micromolecular compound adjusting DNA and transcribe, PPARs can regulate and control the destination gene expression of the inside and outside lipid metabolism of many participation cells, especially the gene of some important enzymes in the beta-oxidation process of encoding, PPARs also participates in differentiation (the Evans RM of adipocyte in addition, Barish GD, Wang YX.PPARs and the complex journey to obesity.Nat Med.2004,10:355-361.).
Summary of the invention
The method and the special product thereof that the purpose of this invention is to provide a kind of assistant identification fat thickness at back of pig proterties.
Assistant identification provided by the invention detects the method for fat thickness at back of pig proterties, be that the GenBank Accession Number GU565976.1(update date that detects peroxisome proliferation-activated receptors δ (PPARD) gene of pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or T and C, genotype with definite described pig to be measured is TT or TC or CC, determine thickness of backfat proterties according to the genotype of described pig to be measured: the thickness of backfat of the genotypic pig to be measured of TT is higher than or the candidate is higher than the genotypic pig to be measured of TC, and the thickness of backfat of the genotypic pig to be measured of TC is higher than or the candidate is higher than the genotypic pig to be measured of CC; Described TT genotype is that the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the homozygote of T; Described CC genotype is that the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the homozygote of C, described TC genotype is that the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the heterozygote of C and T.
In the aforesaid method, the GenBank Accession Number GU565976.1(update date of the peroxisome proliferation-activated receptors δ gene of described detection pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be that specifically to can be the 95th of detecting sequence 3 in the sequence table or sequence 4 be T or C or T and C for T or C or T and C.
In the aforesaid method, the GenBank Accession Number GU565976.1(update date of the peroxisome proliferation-activated receptors δ gene of described detection pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or the method for T and C specifically can be sequencing analysis; Describedly state that sequencing analysis comprises pcr amplification and to pcr amplification product two steps that check order; The used primer of described pcr amplification is to satisfying following condition: be that the GenBank Accession Number GU565976.1(update date that product that template is carried out pcr amplification contains the peroxisome proliferation-activated receptors δ gene of pig is on April 26th, 2010 with the genomic dna of pig to be measured) the 47033rd deoxyribonucleotide.
In the aforesaid method, the used primer of described pcr amplification is to specifically can be right for the primer of being made up of the single stranded DNA shown in the sequence 2 in the single stranded DNA shown in the sequence in the sequence table 1 and the sequence table.
A kind of special product of assistant identification fat thickness at back of pig proterties provided by the present invention is the reagent of the thickness of backfat proterties of assistant identification pig.
The reagent of the thickness of backfat proterties of assistant identification pig provided by the present invention is that the GenBank Accession Number GU565976.1(update date that detects the peroxisome proliferation-activated receptors δ gene of pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or the material of T and C.
In the mentioned reagent, described material specifically can be right for the primer of being made up of the strand Nucleotide shown in the sequence 2 in the strand Nucleotide shown in the sequence in the sequence table 1 and the sequence table.When adopting described primer when carrying out pcr amplification, according to the difference of the genomic dna of pig to be measured, the dna fragmentation that amplification obtains is the Nucleotide shown in the Nucleotide shown in the sequence 3 or the sequence 4.
The another kind of special product of assistant identification fat thickness at back of pig proterties provided by the present invention is the test kit of reagent that contains the thickness of backfat proterties of above-mentioned assistant identification pig.
Above, the GenBank Accession Number GU565976.1(update date that detects the peroxisome proliferation-activated receptors δ gene of pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or the material of T and C can be by following at least a method determine that the GenBank Accession Number GU565976.1(update date of the peroxisome proliferation-activated receptors δ gene of pig is on April 26th, 2010) the 47033rd required reagent and/or the instrument of single nucleotide polymorphism: dna sequencing, the restriction fragment length polymorphism, single strand conformation polymorphism, sex change high performance liquid chromatography and SNP chip.Wherein, the SNP chip comprises chip based on nucleic acid hybridization reaction, based on the chip of single base extension, based on the chip of allele-specific primers extension, based on the chip of " single stage method " reaction, based on the chip of primer ligation, based on the chip of restriction enzyme reaction, based on the chip of protein D NA association reaction, and based on the chip of fluorescence molecule DNA association reaction.
Application and the application in the product of the thickness of backfat proterties for preparing the assistant identification pig in the thickness of backfat proterties of assistant identification pig of mentioned reagent or mentioned reagent box all belongs to protection scope of the present invention.
Described product can be reagent or test kit, also can be the combined prod of reagent or test kit and instrument, as the combined prod of being formed by primer and dna sequencing instrument, or the combined prod of being formed by PCR reagent and dna sequencing reagent and dna sequencing instrument.
Above, the described thickness of backfat can be the thickness of backfat and/or the average thickness of backfat of the six or seven rib corresponding position.
In an embodiment of the invention, described pig to be measured is big people's hybridized pig.
Aforesaid method, reagent or test kit all can be used for cultivating the pig with thicker or thinner thickness of backfat proterties, thereby are applied to the breeding of pig.
The present invention also provides a kind of method of cultivating thin back fat pig.
The method of the thin back fat pig of cultivation provided by the present invention comprises and selects CC genotype or the genotypic pig of TC to carry out breeding; Described CC genotype is that the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ (PPARD) gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the homozygote of C, described TC genotype is that the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the heterozygote of C and T.
Experimental results show that, CC genotype colony respectively than TT genotype colony in six or seven ribbed back fat thickness and the average thickness of backfat thin 5.49mm and 5.60mm(P＜0.01 respectively), CC genotype colony respectively than TC genotype colony in six or seven ribbed back fat thickness and the average thickness of backfat thin 3.12mm and 3.31mm(P＜0.01 respectively).So the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) the 47033rd can be used as fat thickness at back of pig trait molecular breeding mark.
Use the present invention pig is carried out breeding, can carry out early screening to pig to be selected, alleviate the problem of selecting good boar time length in the actual production effectively, reduce the breeding cost, effectively reduce or improve the fat thickness at back of pig in the actual production.Detection method of the present invention is simple to operate, expense is not high, accuracy is high, and can realize automatic direct detection.
Description of drawings
Fig. 1 is the sequencing result figure (pig PPARD gene g.47033C〉T mutational site sequencer map) of sequence M1 and sequence M2.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, the embodiment that provides is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Big people's hybridized pig among the following embodiment is the F2 generation of Large White * people pig, obtain according to the method in the document: Luo W, Cheng D, Chen S, Wang L, Li Y, Ma X, Song X, Liu X, Li W, Liang J, Yan H, Zhao K, Wang C, Wang L, Zhang L.Genome-wide association analysis of meat quality traits in a porcine Large White * Minzhu intercross population.Int J Biol Sci.2012,8:580-595.Concrete grammar is as follows: the four-head Large White is 16 people pigs of mating respectively, obtain F1 for colony, and this F1 is for 46 sows of 9 boar mating in the colony, and three parity have been produced 575 F2 altogether for individuality.
The thickness of backfat proterties of embodiment 1, assistant identification pig
One, pig PPARD gene is g.47033C〉the determining of T pleomorphism site
1, pcr amplification
According to pig PPARD gene genomic dna sequence (GenBank Accession Number GU565976.1) information, it is as follows to design a pair of primer:
The U(upstream primer): 5 '-caaaacgcagtagcaggaaa-3 ' (sequence 1 of sequence table);
The D(downstream primer): 5 '-cccaacctctctagcacacc-3 ' (sequence 2 of sequence table).
Selecting 3 big people's hybridized pigs is experiment material.Genomic dna with every big people's hybridized pig is template, uses primer U and D to carry out pcr amplification.
Amplification system is: genomic dna 200ng, 10 * pcr amplification damping fluid, 5 μ l, dNTPs10mM, each 50ng of upstream and downstream primer, Taq archaeal dna polymerase 0.75U, Mg
2+2.5mmol/L, use ddH
2O postreaction system to 50 μ l.
The PCR reaction conditions is: 95 ℃ of sex change 5min; 95 ℃ of sex change 20s then, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 35 circulations; Last 72 ℃ are extended 10min.
The PCR product after sepharose detects in-20 ℃ of preservations.Genomic dna with 3 big people's hybridized pigs is product difference called after product 1, product 2, the product 3 that template increases respectively.
2, cloning and sequencing and sequential analysis
Respectively 3 kinds of PCR products are reclaimed test kit (day root biochemical technology company limited) with sepharose and reclaim purifying, behind the dna fragmentation connection carrier pGEM-T (Promega company) that reclaims, to connect product transformed into escherichia coli DH5 α competent cell (proud (Beijing) Science and Technology Ltd. tomorrow hundred), carboxylic Bian penicillin resistance label screening positive colony according on the carrier obtains containing the recombinant plasmid that reclaims fragment.Being primer with the T7 on this recombinant plasmid vector and SP6 promoter sequence carries out nucleotide sequencing (the prompt base in the English Weihe River (Shanghai) trade Co., Ltd) to it.Sequencing result shows: product 1 is sequence M1, and product 2 is the mixture of sequence M1 and sequence M2, and product 3 is sequence M2; The length of sequence M1 and sequence M2 is 212bp, difference (T/C) (see figure 1) that only has a deoxyribonucleotide, this deoxyribonucleotide is that GenBank Accession Number GU565976.1(update date is on April 26th, 2010) from 5 ' terminal the 47033rd deoxyribonucleotide, with this mononucleotide polymorphic called after g.47033C〉T.The nucleotide sequence of sequence M1 is seen the sequence 3 of sequence table, and the nucleotide sequence of sequence M2 is seen the sequence 4 of sequence table.Be on April 26th, 2010 with the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene) from 5 ' terminal the 47033rd homozygote genotype called after TT that deoxyribonucleotide is T, the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ gene is on April 26th, 2010) from 5 ' homozygote genotype that terminal the 47033rd deoxyribonucleotide is C is CC, their heterozygote genotype is TC.
Two, pig PPARD gene is g.47033C〉correlation analysis of T polymorphic site and fat thickness at back of pig and meat matter proterties
For determining g.47033C〉whether the T polymorphic site relevant with the fat thickness at back of pig proterties, be experiment material with 575 big people's hybridized pigs, carry out following test: the genomic dna that extracts every pig, carry out pcr amplification and the pcr amplification product of every pig is carried out sequencing analysis with above-mentioned primer U and D, the genotype of determining every pig is TT or TC or CC, the same step 1 of method.Determine thickness of backfat proterties according to the genotype of pig: the thickness of backfat of the genotypic pig to be measured of TT is higher than the genotypic pig to be measured of TC, and the thickness of backfat of the genotypic pig to be measured of TC is higher than the genotypic pig to be measured of CC.
Measure and the thickness of backfat (thickness of backfat of shoulder thickness fat thickness, the six or seven rib corresponding position (six or seven ribbed back fat thickness), chest lumbar fusion place fat thickness, waist are recommended vertebra joint fat thickness), intramuscular fat content proterties such as (IMF) when recording every pig 240 ages in days, to g.47033C T site and proterties carry out association analysis with method of least squares.Used model is as follows:
Y=S+P+B+G+e
Wherein Y is the property determination value, and S is the sex effect, and P is the parity effect, and B is for butchering a batch effect, and G is the genotype effect, and e is the residual error effect.
Table 1PPARD gene mutation site is g.47033C〉the T genotype is related with the fatty deposits correlated character
| Genotype | Quantity | Six or seven ribbed back fat thickness (mm) | The average thickness of backfat (mm) | Intramuscular fat (%) |
| TT | 161 | 40.01±0.77 A | 40.14±0.76 A | 3.14±0.18 A |
| TC | 273 | 37.64±0.68 B | 37.85±0.67 B | 3.11±0.16 A |
| CC | 141 | 34.52±0.77 C | 34.54±0.76 C | 3.13±0.18 A |
Annotate: same column subscript letter identical table differential different not significantly (P〉0.01), same column subscript letter different table differential heteropole is (P＜0.01) significantly.
Wherein, fat thickness refers to the thickness of subcutaneous lipids, does not contain skin depth.Get fat thickness, chest lumbar fusion place fat thickness, the waist of shoulder thickness fat thickness, six or seven rib corresponding positions and recommend the mean value of vertebra joint fat thickness as the average thickness of backfat.
Result such as table 1 show that in the thickness of backfat proterties thickness of backfat of TT genotype pig is higher than TC genotype pig, and the genotypic fat thickness at back of pig of TC is higher than the genotypic pig of CC; CC genotype colony is respectively than the thickness of backfat and average thickness of backfat difference thin 5.49mm and 5.60mm(P＜0.01 of TT genotype colony in six or seven rib corresponding positions), CC genotype colony is respectively than the thickness of backfat and average thickness of backfat difference thin 3.12mm and 3.31mm(P＜0.01 of TC genotype colony in six or seven rib corresponding positions).Illustrate that it is on April 26th, 2010 that the present invention utilizes the GenBank Accession Number GU565976.1(update date of peroxisome proliferation-activated receptors δ (PPARD) gene) the 47033rd single nucleotide polymorphism identify that the method for fat thickness at back of pig proterties is consistent with the practical measurement result of fat thickness at back of pig.
So, in the pig breeding of reality, for obtaining the pig of thinner back fat, preferably select the genotypic pig of CC to carry out breeding.
Claims (10)
1. the method for the thickness of backfat proterties of an assistant identification pig, be that the 47033rd deoxyribonucleotide of GenBank Accession Number GU565976.1 that detects the peroxisome proliferation-activated receptors δ gene of pig to be measured is T or C or T and C, genotype with definite described pig to be measured is TT or TC or CC, determine thickness of backfat proterties according to the genotype of described pig to be measured: the thickness of backfat of the genotypic pig to be measured of TT is higher than or the candidate is higher than the genotypic pig to be measured of TC, and the thickness of backfat of the genotypic pig to be measured of TC is higher than or the candidate is higher than the genotypic pig to be measured of CC; Described TT genotype be peroxisome proliferation-activated receptors δ gene GenBank Accession Number GU565976.1 from 5 ' terminal the 47033rd deoxyribonucleotide is the homozygote of T; Described CC genotype be peroxisome proliferation-activated receptors δ gene GenBank Accession Number GU565976.1 from 5 ' terminal the 47033rd deoxyribonucleotide is the homozygote of C, described TC genotype be peroxisome proliferation-activated receptors δ gene GenBank Accession Number GU565976.1 be the heterozygote of C and T from 5 ' terminal the 47033rd deoxyribonucleotide.
2. method according to claim 1 is characterized in that: the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the peroxisome proliferation-activated receptors δ gene of described detection pig to be measured is T or C or the method for T and C is sequencing analysis; Describedly state that sequencing analysis comprises pcr amplification and to pcr amplification product two steps that check order; The used primer of described pcr amplification is to satisfying following condition: be the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of product that template the is carried out pcr amplification peroxisome proliferation-activated receptors δ gene that contains pig with the genomic dna of pig to be measured.
3. method according to claim 2 is characterized in that: the used primer of described pcr amplification to for the primer of being formed by the single stranded DNA shown in the sequence 2 in the single stranded DNA shown in the sequence in the sequence table 1 and the sequence table right.
4. the reagent of the thickness of backfat proterties of an assistant identification pig is that the 47033rd deoxyribonucleotide of GenBank Accession Number GU565976.1 that detects the peroxisome proliferation-activated receptors δ gene of pig to be measured is T or C or the material of T and C.
5. reagent according to claim 4 is characterized in that: described material is right for the primer of being made up of the DNA shown in the sequence 2 in the DNA shown in the sequence in the sequence table 1 and the sequence table.
6. the test kit of the thickness of backfat proterties of assistant identification pig contains claim 4 or 5 described reagent.
7. claim 4 or 5 described reagent or the described test kit of claim 6 application, the application in the product of the thickness of backfat proterties for preparing the assistant identification pig in the thickness of backfat proterties of assistant identification pig.
8. according to arbitrary described among the claim 1-7, it is characterized in that: the described thickness of backfat is the thickness of backfat and/or the average thickness of backfat of six or seven rib corresponding positions.
9. the described method of claim 1-8, reagent or test kit are in Application in pig Breeding.
10. cultivate the method for thin back fat pig, comprise and select CC genotype or the genotypic pig of TC to carry out breeding; Described CC genotype be peroxisome proliferation-activated receptors δ gene GenBank Accession Number GU565976.1 from 5 ' terminal the 47033rd deoxyribonucleotide is the homozygote of C, described TC genotype be peroxisome proliferation-activated receptors δ gene GenBank Accession Number GU565976.1 be the heterozygote of C and T from 5 ' terminal the 47033rd deoxyribonucleotide.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195136A (en) * | 2014-09-04 | 2014-12-10 | 中国农业科学院北京畜牧兽医研究所 | Method or identifying length and/or height of pig body and special primer pair therefor |
| CN105525007A (en) * | 2016-01-27 | 2016-04-27 | 中国农业科学院北京畜牧兽医研究所 | Living pig backfat thickness marker assisted selection method based on SMAD7 gene |
| CN107151692A (en) * | 2016-03-02 | 2017-09-12 | 中国农业科学院北京畜牧兽医研究所 | A kind of gene diagnosis kit for identifying the pig live body thickness of backfat |
| CN112126691A (en) * | 2020-10-15 | 2020-12-25 | 上海新农科技股份有限公司 | Method, primer, kit and application for auxiliary detection of pig backfat thickness character by utilizing pig No. 2 chromosome gene polymorphic site |
| CN113373142A (en) * | 2020-03-09 | 2021-09-10 | 中国农业科学院深圳农业基因组研究所 | Molecular marker-assisted selection method for pig backfat thickness and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195136A (en) * | 2014-09-04 | 2014-12-10 | 中国农业科学院北京畜牧兽医研究所 | Method or identifying length and/or height of pig body and special primer pair therefor |
| CN105525007A (en) * | 2016-01-27 | 2016-04-27 | 中国农业科学院北京畜牧兽医研究所 | Living pig backfat thickness marker assisted selection method based on SMAD7 gene |
| CN107151692A (en) * | 2016-03-02 | 2017-09-12 | 中国农业科学院北京畜牧兽医研究所 | A kind of gene diagnosis kit for identifying the pig live body thickness of backfat |
| CN107151692B (en) * | 2016-03-02 | 2020-05-22 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying pig living body backfat thickness and gene diagnosis kit |
| CN113373142A (en) * | 2020-03-09 | 2021-09-10 | 中国农业科学院深圳农业基因组研究所 | Molecular marker-assisted selection method for pig backfat thickness and application thereof |
| CN113373142B (en) * | 2020-03-09 | 2023-02-21 | 中国农业科学院深圳农业基因组研究所 | Molecular marker-assisted selection method for pig backfat thickness and application thereof |
| CN112126691A (en) * | 2020-10-15 | 2020-12-25 | 上海新农科技股份有限公司 | Method, primer, kit and application for auxiliary detection of pig backfat thickness character by utilizing pig No. 2 chromosome gene polymorphic site |
| CN112126691B (en) * | 2020-10-15 | 2024-04-26 | 上海新农科技股份有限公司 | Method, primer, kit and application for auxiliary detection of pig backfat thickness character by using pig chromosome 2 gene polymorphism site |
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