CN101812450B - Auxiliary identification method of chickens with different weight characters and special primers thereof - Google Patents
Auxiliary identification method of chickens with different weight characters and special primers thereof Download PDFInfo
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- CN101812450B CN101812450B CN2010101612350A CN201010161235A CN101812450B CN 101812450 B CN101812450 B CN 101812450B CN 2010101612350 A CN2010101612350 A CN 2010101612350A CN 201010161235 A CN201010161235 A CN 201010161235A CN 101812450 B CN101812450 B CN 101812450B
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Abstract
The invention discloses an auxiliary identification method of chickens with different weight characters and special primers thereof. The special primers refer to a primer pair consisting of DNA shown as sequences 3 and 4 and the primer can be used for the auxiliary identification of chickens with different weight characteristics. The method comprises the steps of carrying out PCR (Polymerase Chain Reaction) by using the primer pair by taking gene group DNA of a chicken to be tested as a template; if no 336bp PCR product but a 398bp PCR product is obtained, judging the gene type of the chickenis AA; if no 398bp PCR product but the 336bp PCR product is obtained, judging the gene type of the chicken is BB; and if both the 398bp PCR product and the 336bp PCR product are obtained, judging the gene type of the chicken is AB. A chicken of AA gene type is heavier than a chicken of AB gene type in statistics and a chicken of AB gene type is heavier than a chicken of BB gene type in statistics. The method has the advantages of simpleness, convenience, quickness, high sensitivity, reliable, stable and accurate result and no need of special instruments.
Description
Technical field
The present invention relates to method and primer special thereof that a kind of assistant identification has the different weight characters chicken.
Background technology
Transforming growth factor superfamily (Transforming growth factor B) is one type of structurally conservative relatively growth factor family; Participate in various kinds of cell physiological activities such as cell proliferation and differentiation, all play an important role at the aspects such as differentiation, increment and immunomodulatory of fetal development, the generation of histoorgan form, cell.TGFB2 is one of TGFB family member, and being influences the endogenic negative regulation growth factor that animal body grows.So the variation of TGFB2 gene has remarkable influence to the body weight of animal.Chicken TGFB2 gene is positioned in about the terminal 70cM in No. 3 karyomit(e) kinetochores, total length 70kp, and by 7 exons, 6 introns are formed.
In recent years, many researchs were positioned near TGFB2 gene region or its zone influencing the relevant QTLs of body weight.Kerje etc. (2003) are with a F
2The resource family is a material, the QTLs that finds to influence chicken 8 age in days body weight and 46 age in days body weight at No. 3 chromosomal 50cM places and the 117cM place of chicken respectively.Ambo etc. (2009) discover that the QTLs that influences 35 and 41 age in days weight characters is positioned at chromosomal 50-200cM place No. 3.Therefore, the TGFB2 gene is expected to become the candidate gene of chicken weight character, is applied in the chicken molecular breeding as genetic marker.
Polymerase chain reaction (PCR) technology is a kind of specific DNA amplification in vitro technology.With a spot of DNA is template, present with a large amount of target dna molecule of form generation near the index amplification through the repeatedly circulation of sex change-annealing-extensions, and that this technology has become is the most frequently used, also one of most important Protocols in Molecular Biology.The PCR product can carry out genotypic evaluation work through after the agarose electrophoresis, and detection method is simple.
Summary of the invention
The purpose of this invention is to provide method and primer special thereof that a kind of assistant identification has the different weight characters chicken.
It is right to the invention provides the primer that DNA forms shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
Said primer has the chicken of different weight characters to can be applicable to assistant identification.
Said primer is to can be applicable to prepare the test kit that assistant identification has the different weight characters chicken.In the test kit, also can comprise other pcr analysis reagent.Like archaeal dna polymerase, PCR damping fluid, dATP, dGTP, dCTP, dTTP etc., these pcr analysis reagent all can be existing conventional reagent.
The present invention also protects assistant identification to have the test kit of different weight characters chicken, comprises that said primer is right.
Assistant identification provided by the invention has the method for different weight characters chicken, comprises the steps (a) or step (b):
(a) genomic dna with chicken to be measured is a template, and to carrying out pcr amplification, the body weight that does not obtain the chicken of 336bpPCR product is higher than the body weight of the chicken that obtains 336bp PCR product on statistics with said primer;
(b) detect dna fragmentation shown in the sequence 2 that whether has sequence table in the genomic dna of chicken to be measured; The body weight that does not have the chicken of dna fragmentation shown in the sequence 2 in the genomic dna is higher than the body weight that has the chicken of dna fragmentation shown in the sequence 2 in the genomic dna on statistics.
Assistant identification provided by the invention has the method for different weight characters chicken, comprises the steps (a) or step (b):
(a) genomic dna with chicken to be measured is a template, and to carrying out pcr amplification, the body weight that obtains the chicken of 398bp PCR product is higher than the body weight of the chicken that does not obtain 398bp PCR product on statistics with said primer;
(b) detect dna fragmentation shown in the sequence 1 that whether has sequence table in the genomic dna of chicken to be measured; There is the body weight of the chicken of dna fragmentation shown in the sequence 1 on statistics, to be higher than the body weight that does not have the chicken of dna fragmentation shown in the sequence 1 in the genomic dna in the genomic dna.
Assistant identification provided by the invention has the method for different weight characters chicken, comprises the steps (a) or step (b):
(a) genomic dna with chicken to be measured is a template, with said primer to carrying out pcr amplification; If obtain the 398bpPCR product and do not obtain 336bp PCR product, the genotype of chicken to be measured is AA; If obtain 336bp PCR product and do not obtain 398bp PCR product, the genotype of chicken to be measured is BB; If obtain two kinds of PCR products of 398bp and 336bp, the genotype of chicken to be measured is AB; The body weight of AA genotype chicken is higher than the body weight of AB genotype chicken on statistics; The body weight of AB genotype chicken is higher than the body weight of BB genotype chicken on statistics;
(b) detect dna fragmentation shown in the sequence 2 of dna fragmentation shown in the sequence 1 that whether has sequence table in the genomic dna of chicken to be measured and sequence table; Be the homozygote of dna fragmentation shown in the sequence 1 in the genomic dna, genotype is AA; Be the homozygote of dna fragmentation shown in the sequence 2 in the genomic dna, genotype is BB; Be the heterozygote of sequence 1 and dna fragmentation shown in the sequence 2 in the genomic dna, genotype is AB; The body weight of AA genotype chicken is higher than the body weight of AB genotype chicken on statistics; The body weight of AB genotype chicken is higher than the body weight of BB genotype chicken on statistics.
Assistant identification provided by the invention has the method for different weight characters chicken, comprises the steps: to detect respectively in the genomic dna of every chicken to be measured whether have specific DNA fragments; There is the mean body weight of the chicken colony of specific DNA fragments on statistics, to be higher than the mean body weight of the chicken colony that does not have specific DNA fragments in the genomic dna in the genomic dna; Said specific DNA fragments is that the sequence 1 of sequence table is held dna fragmentation shown in the 37th to 98 Nucleotide from 5 '.
More than in arbitrary described method, said chicken to be measured specifically can be Beijing fine breed of chicken with thick brownish feathers.
More than in arbitrary described method, said chicken to be measured specifically can be hen.
More than in arbitrary described method, said chicken to be measured specifically can be into chicken.Said chicken to be measured specifically can be chicken, the chicken in 11 ages in week, the chicken in 13 ages in week or the chicken in 17 ages in week in 7 ages in week.
Above method, test kit, primer are to all can be applicable to a breed of chicken.
The present invention is based on and detect chicken TGFB2 gene g.-851_-790del polymorphic site, provide method that assistant identification has the different weight characters chicken, primer to and test kit.Method of the present invention has following advantage: easy, quick, sensitive, reliable results, stable, accurate does not need specific apparatus, is fit to the needs that the routine test chamber is detected.
Description of drawings
Fig. 1 is the PCR product electrophorogram of each genotypic sample.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
To the TGFB2 gene of the several body of Beijing fine breed of chicken with thick brownish feathers (the GenBank accession number: X58071) and the neighboring area detect.Find that in the part individuality, the deletion mutantion (g.-851_-790del) of a 62bp has taken place to-790 places transcription initiation site-851 in the TGFB2 gene promoter area, thereby has influenced the TGFB2 expression of gene.
According to sudden change zone design primer to as follows:
Upstream primer (F): 5 '-GGAGGAAAAGAGTGGAGTGCT-3 ' (sequence 3);
Downstream primer (R): 5 '-CTGCGTCTGCAATTCAGTTT-3 ' (sequence 4).
Primer is synthetic by Beijing AudioCodes biotech firm.
Test uses chicken as Beijing fine breed of chicken with thick brownish feathers hen (totally 1030), is given by Institute of Animal Sciences, Chinese Academy of Agricultural Sciences test base, raises in national poultry and measures the center, and China Agricultural University guarantees to provide to the public.Reference: Liu Chen; Wen Jie; Zhao Guiping; Zheng Maiqing; Chen Jilan; A-FABP and AMPD1 gene SNP site are to combined effect analysis, the journal of animal science and veterinary medicine of Beijing fine breed of chicken with thick brownish feathers part meat proterties, 2009,40 (10): 1555-1559.
One, phenotype record
Measure individual growth traits phenotypic number: 7,11,13,17 age in week body weight.7, be early stage body weight 11,13 ages in week.17 the week age body weight be adult weight.
Two, genotype detection
1, blood specimen collection
During 9 ages in week, wing blood sampling down adds the ACD anti-freezing ,-20 ℃ of preservations.
2, the extraction of poba gene group DNA:
Adopt the poba gene group DNA extraction test kit DP318 of TIANGEN Biotech (Beijing) Co., Ltd. to carry out the extraction of poba gene group DNA.
3, pcr amplification
The genomic dna that extracts with step 2 is a template, and is right as primer with two primers (sequence 3 and sequence 4) of embodiment 1 preparation, carries out pcr amplification.
Pcr amplification reaction system (25 μ l): dna profiling (100ng/ μ l) 1 μ l; Article two, each 0.6 μ l of primer, final concentration is 10pmol/ μ l; DNTP miscellany 2.0 μ l, the concentration of four kinds of dNTP is 2.5mmol/L; TaqDNA polysaccharase (5U/ μ l) 0.2 μ l; 10 * PCR damping fluid (Mg
2+Free) 2.5 μ l; 25mmol/l MgCl
21.5ul; Add ultrapure water to 25ul.
Pcr amplification condition: 95 ℃ of preparatory sex change 5min of elder generation; 95 ℃ of 30s then, 61 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; Last 72 ℃ are extended 10min.
4, order-checking
PCR product with step 3 checks order respectively.The result shows that sample evidence PCR product can be divided into three kinds:
First kind: the PCR product is a dna fragmentation (398bp), shown in the sequence 1 of sequence table; With sample genotype called after insert type homozygote (AA genotype);
Second kind: the PCR product is a dna fragmentation (336bp), shown in the sequence 2 of sequence table; With sample genotype called after absence type homozygote (BB genotype);
The third: the PCR product is two dna fragmentations (398bp and 336bp), respectively shown in the sequence 2 of the sequence 1 of the sequence table of sequence table and sequence table; With sample genotype called after heterozygote (AB genotype);
5, agarose gel electrophoresis
The pcr amplification product of step 3 is carried out 3% agarose gel electrophoresis, and the result sees Fig. 1.Among Fig. 1: swimming lane M:DNA molecular weight standard Marker I (day root); Swimming lane 2,5,7,8,9,11: insert type homozygote; Swimming lane 1,4,6,10: heterozygote; Swimming lane 3: absence type homozygote.
Agarose gel electrophoresis is the result show, the insert type homozygote is 647 in these 1030 Beijing fine breed of chicken with thick brownish feathers, 356 of heterozygotes, 27 of absence type homozygotes.
Three, association analysis
The MIXED process that adopts SASV9.2 software to 7,11,13,17 age in week body weight and genotype carry out association analysis, linear equation is following:
y＝μ+S+D(S)+G+e；
Wherein: y=growth traits (7,11,13,17 week age body weight observed value)
μ=colony's average
S=cock effect (stochastic effect)
Hen effect (stochastic effect) in the D=cock
G=genotype effect (fixed effect)
E=random residual effect
Additive effect (a) and dominant effect (d) calculation formula are:
a＝(AA-BB)/2；d＝AB-(AA+BB)/2。
The Bofferoni The result of multiple comparisons is seen table 1, and the result of association analysis sees table 2.
Table 1Bofferoni The result of multiple comparisons
Annotate: A, B represent difference extremely significantly (P＜0.01); A, b represent significant difference (0.01＜P＜0.05).
Table 2g.-851_-790del genotype association analysis result
| |
7 all body weight (g) | 11 all body weight (g) | 13 all body weight (g) | 17 all body weight (g) |
| The P value | 0.0013 ** | 0.0369 * | 0.0241 * | 0.0035 ** |
| Additive effect | 8.85±6.63 | 23.93±15.30 | 14.87±12.24 | 45.45±15.54 ** |
| Dominant effect | -9.47±7.54 | -2.55±17.40 | -9.88±13.92 | 19.36±17.67 |
Annotate:
*Expression difference is (P＜0.01) extremely significantly;
*Expression significant difference (0.01＜P＜0.05).
Association analysis is the result show, TGFB2 gene g.-851_-790del polymorphic site to age in Beijing fine breed of chicken with thick brownish feathers 7,17 week body weight reach extremely significantly (p＜0.01) level, to 11,13 age in week body weight reach level of signification (p＜0.05).Bonferroni multiple comparisons analytical proof the homozygous mean body weight of TGFB2 insert type be significantly higher than heterozygote and the genotypic mean body weight of absence type homozygote (P＜0.05).After 11 ages in week, the mean body weight of heterozygote is significantly higher than the genotypic mean body weight of absence type homozygote.The allelic substitution effect shows, 17 age in week body weight additive effect reached extremely significant level (P＜0.01), the effect that the g.-851_-790del site is described is heritable.
Sequence table
< 110>China Agricultural University
< 120>assistant identification has method and the primer special thereof of different weight characters chicken
<130>CGGNARY102260
<160>4
<210>1
<211>398
<212>DNA
< 213>chicken (Gallus domestiaus)
<400>1
ggaggaaaag?agtggagtgc?ttttacagag?ttattaaggt?tggaaaagat?gactgtgatc 60
atttagtagt?ccaacccttg?agggctgtga?agccccccag?gcaagtgatg?ggcagtgcag 120
cacgggcagc?aaccacgcgg?cgtggggcca?taggttcagt?gcaaggcatt?tctccatggt 180
ttcagctgca?gctttaggag?gaaatcactc?aatagccttt?ttcttcggaa?gagtttgctt 240
tacggtacaa?ggctgagggg?gtgtgcaagg?tcatttctgt?agggcagagc?ggggcccgag 300
gtcagagctc?ggcctttgcc?tgcacacggc?tccgggctca?ggctgcgatg?tgcgctgcgg 360
ccgaaagagg?cagtggaaaa?actgaattgc?agacgcag 398
<210>2
<211>336
<212>DNA
< 213>chicken (Gallus domestiaus)
<400>2
ggaggaaaag?agtggagtgc?ttttacagag?ttattaaggc?aagtgatggg?cagtgcagca 60
cgggcagcaa?ccacgcggcg?tggggccata?ggttcagtgc?aaggcatttc?tccatggttt 120
cagctgcagc?tttaggagga?aatcactcaa?tagccttttt?cttcggaaga?gtttgcttta 180
cggtacaagg?ctgagggggt?gtgcaaggtc?atttctgtag?ggcagagcgg?ggcccgaggt 240
cagagctcgg?cctttgcctg?cacacggctc?cgggctcagg?ctgcgatgtg?cgctgcggcc 300
gaaagaggca?gtggaaaaac?tgaattgcag?acgcag 336
<210>3
<211>21
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
ggaggaaaag?agtggagtgc?t 21
<210>4
<211>20
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
ctgcgtctgc?aattcagttt 20
Claims (8)
1. the primer of the composition of DNA shown in the sequence 4 of DNA and sequence table shown in the sequence 3 of sequence table is right.
2. the said primer of claim 1 is to having the application in the different weight characters chicken in assistant identification; Said chicken is Beijing fine breed of chicken with thick brownish feathers.
3. the said primer of claim 1 is to the application in the test kit that has the different weight characters chicken in the preparation assistant identification; Said chicken is Beijing fine breed of chicken with thick brownish feathers.
4. an assistant identification has the test kit of different weight characters chicken, comprises that the said primer of claim 1 is right; Said chicken is Beijing fine breed of chicken with thick brownish feathers.
5. an assistant identification has the method for different weight characters chicken; Comprise the steps: that the genomic dna with chicken to be measured is a template; To carrying out pcr amplification, the body weight that does not obtain the chicken of 336bp PCR product is higher than the body weight of the chicken that obtains 336bp PCR product on statistics with the said primer of claim 1; Said chicken to be measured is Beijing fine breed of chicken with thick brownish feathers.
6. an assistant identification has the method for different weight characters chicken; Comprise the steps: that the genomic dna with chicken to be measured is a template; To carrying out pcr amplification, the body weight that obtains the chicken of 398bp PCR product is higher than the body weight of the chicken body that does not obtain 398bp PCR product on statistics with the said primer of claim 1; Said chicken to be measured is Beijing fine breed of chicken with thick brownish feathers.
7. an assistant identification has the method for different weight characters chicken, comprises the steps: that the genomic dna with chicken to be measured is a template, with the said primer of claim 1 to carrying out pcr amplification; If obtain 398bp PCR product and do not obtain 336bp PCR product, the genotype of chicken to be measured is AA; If obtain 336bp PCR product and do not obtain 398bp PCR product, the genotype of chicken to be measured is BB; If obtain two kinds of PCR products of 398bp and 336bp, the genotype of chicken to be measured is AB; The body weight of AA genotype chicken is higher than the body weight of AB genotype chicken on statistics; The body weight of AB genotype chicken is higher than the body weight of BB genotype chicken on statistics;
Said chicken to be measured is Beijing fine breed of chicken with thick brownish feathers.
8. like arbitrary described method in the claim 5 to 7, it is characterized in that: said chicken to be measured is chicken, the chicken in 11 ages in week, the chicken in 13 ages in week or the chicken in 17 ages in week in 7 ages in week.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102251051A (en) * | 2011-08-15 | 2011-11-23 | 扬州大学 | Kit for detecting Bian chicken MSTN genotype |
| CN103374629B (en) * | 2012-04-28 | 2014-08-06 | 中国农业科学院北京畜牧兽医研究所 | Method for authenticating Beijing fatty chickens by microsatellite markers |
| CN114622020B (en) * | 2022-03-30 | 2022-09-27 | 华南农业大学 | KLHL31 gene molecular marker related to chicken growth traits and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1162978A (en) * | 1994-08-17 | 1997-10-22 | 洛克菲勒大学 | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
| CN101307358A (en) * | 2008-06-20 | 2008-11-19 | 东北农业大学 | Haploid type marking method for predicting and identifying weight and metatarsal bone trait of chicken |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1162978A (en) * | 1994-08-17 | 1997-10-22 | 洛克菲勒大学 | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
| CN101307358A (en) * | 2008-06-20 | 2008-11-19 | 东北农业大学 | Haploid type marking method for predicting and identifying weight and metatarsal bone trait of chicken |
Non-Patent Citations (1)
| Title |
|---|
| Li H等.Chicken quantitative trait loci for growth and body composition associated with transforming growth factor-beta genes.《Poultry Science》.2003,第82卷(第3期),347-356. * |
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