CN103320516B - 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 large good characteristic that China's local pig breed is well-known is exactly that meat is good, commodity pork is hybridized all because of its good meat in these local variety, the cultivation kind that contains local variety blood or even China and foreign countries, extensively get consumer reception, such as Beijing Black pork, Laiwu Black Pigs meat, Black Hills pork, the too pork etc. of reviving are subject to human consumer's favorable comment deeply.And the 1.5-3 that the price of these high-quality porks is common pork doubly, has fully demonstrated the commercial value of current generation China's local variety pig.But these pig kind majorities are all lard type, and its distinguishing feature is that back-fat thickness is large.Back fat deposition too much not only causes poor growth, does not also meet the requirement of existing situation for joint grain simultaneously.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(of genetic correlation Sun Hua with lean ratio, Song Zhongxu, Li Lianghua, Peng Xianwen, Guo Wanzheng, military Hua Yu, Mei Shuqi. Chinese Large White S II 1 owner wants the genetic parameter estimation of Growth and carcass character. hubei agricultural science .2009, 48, 3086-3089.).Therefore, China's local pig breed and the kind thickness of backfat that contains Native 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 compared with international business pig kind, potentiality are also significantly improved.
Based on the importance of above-mentioned fat thickness at back of pig proterties, carry out for it emphasis that breeding work is current breeding work.Because traditional breeding method speed is slow, order of accuarcy is low, molecular breeding has become current breeding work technical way with features 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 mediate lipotropy micromolecular compound and regulate DNA to 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 object of this invention is to provide a kind of method and special product thereof 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, that the GenBank Accession Number GU565976.1(update date of Peroxisome proliferator-activated receptor δ (PPARD) gene that detects pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or T and C, to determine that the genotype of 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 higher than or candidate higher than the genotypic pig to be measured of TC, the thickness of backfat of the genotypic pig to be measured of TC higher than or candidate higher than the genotypic pig to be measured of CC, the GenBank Accession Number GU565976.1(update date that described TT genotype is Peroxisome proliferator-activated receptor δ gene is on April 26th, 2010) the 47033rd homozygote that deoxyribonucleotide is T, the GenBank Accession Number GU565976.1(update date that described CC genotype is Peroxisome proliferator-activated receptor δ gene is on April 26th, 2010) the 47033rd homozygote that deoxyribonucleotide is C, the GenBank Accession Number GU565976.1(update date that described TC genotype is Peroxisome proliferator-activated receptor δ gene is on April 26th, 2010) the 47033rd deoxyribonucleotide be the heterozygote of C and T.
In aforesaid method, the GenBank Accession Number GU565976.1(update date of the Peroxisome proliferator-activated receptor δ gene of described detection pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be that T or C or T and C specifically can be that to detect sequence 3 in sequence table or sequence 4 the 95th be T or C or T and C.
In aforesaid method, the GenBank Accession Number GU565976.1(update date of the Peroxisome proliferator-activated receptor 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; Described pcr amplification primer pair used meets following condition: the GenBank Accession Number GU565976.1(update date that carries out the Peroxisome proliferator-activated receptor δ gene that the product of pcr amplification contains pig taking the genomic dna of pig to be measured as template is on April 26th, 2010) the 47033rd deoxyribonucleotide.
In aforesaid method, described pcr amplification primer pair used can be specifically the primer pair by the single stranded DNA shown in sequence 2 forms in the single stranded DNA shown in sequence in sequence table 1 and 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 of Peroxisome proliferator-activated receptor δ gene that detects pig to be measured is on April 26th, 2010) the 47033rd deoxyribonucleotide be T or C or the material of T and C.
In mentioned reagent, described material can be specifically the primer pair by the strand Nucleotide shown in sequence 2 forms in the strand Nucleotide shown in sequence in sequence table 1 and sequence table.In the time adopting described primer pair to carry 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 sequence 3 or 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 the reagent of the thickness of backfat proterties that contains above-mentioned assistant identification pig.
Above, the GenBank Accession Number GU565976.1(update date that detects the Peroxisome proliferator-activated receptor 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 one method determine that the GenBank Accession Number GU565976.1(update date of the Peroxisome proliferator-activated receptor δ gene of pig is on April 26th, 2010) required reagent and/or the instrument of single nucleotide polymorphism of the 47033rd: DNA sequencing, Restriction fragment length polymorphism, single strand conformation polymorphism, sex change high performance liquid chromatography and SNP chip.Wherein, SNP chip comprises the chip based on nucleic acid hybridization reaction, the chip based on single base extension, the chip based on allele-specific primers extension, the chip based on " single stage method " reaction, the chip based on primer ligation, the chip based on restriction enzyme reaction, chip based on protein D NA association reaction, and chip based on fluorescence molecule DNA association reaction.
The application in the thickness of backfat proterties of assistant identification pig of mentioned reagent or mentioned reagent box and the application in the product of thickness of backfat proterties of preparing assistant identification pig all belong 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 being formed by primer and DNA sequencer, or the combined prod being formed by PCR reagent and DNA sequencing reagent and DNA sequencer.
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 large people's hybridized pig.
Aforesaid method, reagent or test kit all can be used for cultivation and have the pig of 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 proliferator-activated receptor δ (PPARD) gene is on April 26th, 2010) the 47033rd homozygote that deoxyribonucleotide is C, the GenBank Accession Number GU565976.1(update date that described TC genotype is Peroxisome proliferator-activated receptor 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 at 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 at 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 proliferator-activated receptor δ gene is on April 26th, 2010) the 47033rd can be used as fat thickness at back of pig trait molecular breeding mark.
Application the present invention carries out breeding to pig, can carry out early screening to pig to be selected, effectively alleviates the problem of selecting good boar time length in actual production, reduces breeding cost, effectively reduces or improve the fat thickness at back of pig in actual production.Detection method of the present invention is simple to operate, expense is not high, accuracy is high, and can realize the direct-detection of automatization.
Brief description of the drawings
Fig. 1 is the sequencing result figure (g.47033C>T mutational site sequencer map of pig PPARD gene) of sequence M1 and sequence M2.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Large people's hybridized pig in following embodiment is the F2 generation of Large White × people pig, obtain according to the method in 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: four-head Large White is 16 people pigs of mating respectively, obtains F1 generation colony, 46 sows of 9 boar mating in this F1 generation colony, and three parity have been produced 575 F2 altogether for individuality.
The thickness of backfat proterties of embodiment 1, assistant identification pig
One, g.47033C>T determining of pleomorphism site of pig PPARD gene
1, pcr amplification
According to pig PPARD gene genomic dna sequence (GenBank Accession Number GU565976.1) information, design pair of primers is as follows:
U(upstream primer): 5 '-caaaacgcagtagcaggaaa-3 ' (sequence 1 of sequence table);
D(downstream primer): 5 '-cccaacctctctagcacacc-3 ' (sequence 2 of sequence table).
Selecting 3 large people's hybridized pigs is experiment material.Taking the genomic dna of every large people's hybridized pig as template, use primer U and D to carry out pcr amplification.
Amplification system is: genomic dna 200ng, 10 × pcr amplification damping fluid, 5 μ l, dNTPs10mM, the each 50ng of upstream and downstream primer, Taq archaeal dna polymerase 0.75U, Mg
2+2.5mmol/L, uses ddH
2o postreaction system to 50 μ l.
PCR reaction conditions is: 95 DEG C of sex change 5min; Then 95 DEG C of sex change 20s, 58 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 35 circulations; Last 72 DEG C are extended 10min.
PCR product through sepharose detect after in-20 DEG C of preservations.Product called after product 1, product 2, the product 3 respectively increasing as template taking the genomic dna of 3 large people's hybridized pigs respectively.
2, cloning and sequencing and sequential analysis
Respectively 3 kinds of PCR products are reclaimed to test kit (Tian Gen biochemical technology company limited) with sepharose and reclaim purifying, after the DNA fragmentation connection carrier pGEM-T (Promega company) reclaiming, to connect product and transform bacillus coli DH 5 alpha competent cell (proud (Beijing) Science and Technology Ltd. tomorrow hundred), according to the carboxylic Bian penicillin resistance label screening positive colony on carrier, obtain containing the recombinant plasmid that reclaims fragment.Taking the T7 on this recombinant plasmid vector and SP6 promoter sequence, as primer pair, it carries out nucleotide sequencing (the English Weihe River prompt base (Shanghai) trade Co., Ltd).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, only there is the difference (T/C) (seeing Fig. 1) of a deoxyribonucleotide, this deoxyribonucleotide is that GenBank Accession Number GU565976.1(update date is on April 26th, 2010) from the 47033rd deoxyribonucleotide of 5 ' end, by this mononucleotide polymorphic called after g.47033C>T.The nucleotide sequence of sequence M1 is shown in the sequence 3 of sequence table, and the nucleotide sequence of sequence M2 is shown in the sequence 4 of sequence table.On April 26th, 2010 by the GenBank Accession Number GU565976.1(update date of Peroxisome proliferator-activated receptor δ gene) the homozygote genotype called after TT that is T from the 47033rd deoxyribonucleotide of 5 ' end, the GenBank Accession Number GU565976.1(update date of Peroxisome proliferator-activated receptor δ gene is on April 26th, 2010) the homozygote genotype that is C from the 47033rd deoxyribonucleotide of 5 ' end be CC, their heterozygote genotype is TC.
Two, the g.47033C>T correlation analysis of polymorphic site and fat thickness at back of pig and Meat Quality Traits of pig PPARD gene
For determining that g.47033C>T whether polymorphic site is relevant to fat thickness at back of pig proterties, taking 575 large people's hybridized pigs as experiment material, test as follows: the genomic dna that extracts every pig, carry out pcr amplification and the pcr amplification product of every pig is carried out to 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.
The proterties such as the thickness of backfat (thickness of backfat (six or seven ribbed back fat thickness) of shoulder thickness fat thickness, the six or seven rib corresponding position, chest lumbar fusion place fat thickness, waist are recommended vertebra joint fat thickness), intramuscular fat content (IMF) while measuring and record every pig 240 age in days, to g.47033C>T site and proterties are carried out association analysis with method of least squares.Model used is as follows:
Y=S+P+B+G+e
Wherein Y is property determination value, and S is sex-effects, and P is parity effect, and B is for butchering a batch effect, and G is genotype effect, and e is residual error effect.
G.47033C>T genotype and fatty deposits correlated character associated of table 1PPARD gene mutation site
| 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 |
Note: same column subscript letter identical table differential different not remarkable (P>0.01), different extremely significantly (P<0.01) of differences that represent of same column subscript letter.
Wherein, fat thickness refers to the thickness of subcutaneous lipids, not containing skin depth.Get mean value that fat thickness, chest lumbar fusion place fat thickness, the waist of shoulder thickness fat thickness, six or seven rib corresponding positions recommend vertebra joint fat thickness as the average thickness of backfat.
Result, as table 1, shows in thickness of backfat proterties, and the 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 the thickness of backfat and the average thickness of backfat thin 5.49mm and 5.60mm(P<0.01 respectively in six or seven rib corresponding positions than TT genotype colony respectively), CC genotype colony is the thickness of backfat and the average thickness of backfat thin 3.12mm of difference and 3.31mm(P<0.01 in six or seven rib corresponding positions than TC genotype colony respectively).Illustrate that it is on April 26th, 2010 that the present invention utilizes the GenBank Accession Number GU565976.1(update date of Peroxisome proliferator-activated receptor δ (PPARD) gene) the method for the single nucleotide polymorphism qualification fat thickness at back of pig proterties of the 47033rd consistent with the practical measurement result of fat thickness at back of pig.
So, in actual pig breeding, for obtaining the pig of thinner back fat, preferably select the genotypic pig of CC to carry out breeding.
Claims (14)
1. the method for the thickness of backfat proterties of an assistant identification pig, the 47033rd deoxyribonucleotide that is the GenBank Accession Number GU565976.1 of the Peroxisome proliferator-activated receptor δ gene of detection pig to be measured is T or C or T and C, to determine that the genotype of 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 higher than or candidate higher than the genotypic pig to be measured of TC, the thickness of backfat of the genotypic pig to be measured of TC higher than or candidate higher than the genotypic pig to be measured of CC, the GenBank Accession Number GU565976.1 that described TT genotype is Peroxisome proliferator-activated receptor δ gene from the 47033rd homozygote that deoxyribonucleotide is T of 5 ' end, the GenBank Accession Number GU565976.1 that described CC genotype is Peroxisome proliferator-activated receptor δ gene from the 47033rd homozygote that deoxyribonucleotide is C of 5 ' end, the GenBank Accession Number GU565976.1's that described TC genotype is Peroxisome proliferator-activated receptor δ gene is the heterozygote of C and T from the 47033rd deoxyribonucleotide of 5 ' end.
2. method according to claim 1, is characterized in that: the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the Peroxisome proliferator-activated receptor δ gene of described detection pig to be measured is T or C or the method for T and C is sequencing analysis; Described sequencing analysis comprises pcr amplification and to pcr amplification product two steps that check order; Described pcr amplification primer pair used meets following condition: the 47033rd deoxyribonucleotide that carries out the GenBank Accession Number GU565976.1 of the Peroxisome proliferator-activated receptor δ gene that the product of pcr amplification contains pig taking the genomic dna of pig to be measured as template.
3. method according to claim 2, is characterized in that: described pcr amplification primer pair used is the primer pair by the single stranded DNA shown in sequence 2 forms in the single stranded DNA shown in sequence in sequence table 1 and sequence table.
4. according to arbitrary described method in claim 1-3, 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.
5. a reagent for the thickness of backfat proterties of assistant identification pig is that the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the Peroxisome proliferator-activated receptor δ gene of detection pig to be measured is T or C or the material of T and C.
6. reagent according to claim 5, is characterized in that: described material is the primer pair by the DNA shown in sequence 2 forms in the DNA shown in sequence in sequence table 1 and sequence table.
7. according to the reagent described in claim 5 or 6, 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.
8. the test kit of the thickness of backfat proterties of assistant identification pig, contains reagent claimed in claim 5.
9. test kit according to claim 8, is characterized in that: the test kit of the thickness of backfat proterties of described assistant identification pig contains reagent claimed in claim 6.
10. test kit according to claim 8 or claim 9, 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.
The application of test kit described in reagent or claim 8 or 9 described in 11. claims 5 or 6 in the thickness of backfat proterties of assistant identification pig or the application in the product of thickness of backfat proterties of preparing assistant identification pig.
12. application according to claim 11, 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.
The application of arbitrary described test kit in pig breeding in reagent described in arbitrary in arbitrary described method, claim 5-7 in 13. claim 1-4 or claim 8-10.
14. cultivate the method for thin back fat pig, comprise and select CC genotype or the genotypic pig of TC to carry out breeding; The GenBank Accession Number GU565976.1 that described CC genotype is Peroxisome proliferator-activated receptor δ gene from the 47033rd homozygote that deoxyribonucleotide is C of 5 ' end, the GenBank Accession Number GU565976.1's that described TC genotype is Peroxisome proliferator-activated receptor δ gene is the heterozygote of C and T from the 47033rd deoxyribonucleotide of 5 ' end.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
Families Citing this family (4)
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| CN104195136B (en) * | 2014-09-04 | 2017-01-11 | 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 |
| CN105525007B (en) * | 2016-01-27 | 2019-02-26 | 中国农业科学院北京畜牧兽医研究所 | A kind of pig living body thickness of backfat mark auxiliary selection method based on SMAD7 gene |
| 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 |
| 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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| CN101805739A (en) * | 2009-12-15 | 2010-08-18 | 江西农业大学 | Identification method of PPARD major gene, establishment of molecular breeding method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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