CN104694538A - SNP molecular marker related to chicken polydactyly character and application thereof - Google Patents
SNP molecular marker related to chicken polydactyly character and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker related to chicken polydactyly character and an amplified primer thereof. The molecular marker is chosen from LMBR1 gene, wherein the nucleotide sequence is shown in SEQ ID No.1, the 246bp site is a mutation site, and the basic group is A or C. The invention additionally discloses a method and a kit for detecting the chicken polydactyly genotype. By adopting the detecting method or the kit provided by the invention, whether the Peking soy sauce chicken and the taihe silk chicken carry the polydactyly mutation allelic gene can be detected and the polydactyly characters of the Peking soy sauce chicken and the taihe silk chicken can be determined. The polydactyly character can be kept or rejected as required in the actual producing and breeding process and the rejection effect is complete and accurate.
Description
Technical field
The present invention relates to field of biological detection, specifically, relate to the SNP marker relevant to chicken many toes proterties and application thereof.
Background technology
Beijing Fatty Chicken and Silky fowl, delicious and famous with unique outlook, meat egg, be the excellent local variety of China's preciousness.In natural population, there is many toes proterties in Beijing Fatty Chicken and Silky fowl some individuals, but its many toes proterties is unfixing in colony.In recent years, complicated and changeable due to physical environment, make the frequency of various zoonosis illness outbreak more and more higher, not only cause the injures and deaths of the mankind, inflicted heavy losses on poultry farming simultaneously, under this overall situation, each place government makes laws one after another and cancels live-bird transaction.But the Beijing Fatty Chicken after butchering and Silky fowl are difficult to from making a distinction with other yellow chickens and black chicken in appearance, the person that causes avian production and the very large financial loss of processor.Therefore; many toes proterties is also fixed up by breeding technique by the mechanism that the many toes proterties studying Beijing Fatty Chicken and Silky fowl produces in Beijing Fatty Chicken and Silky fowl colony; carcass is provided to identify as Beijing Fatty Chicken and Silky fowl high-quality chicken kind chicken with for commercial chicken; fundamentally protect intellecture property and the consumer's interests of breeding units, this has very great economic implications and social effect.
Find according to (1920) researchs such as Bond C J, chicken many toes proterties controls by the euchromosome of Incomplete dominance, and traditional breeding way is difficult to many toes proterties in fixing chicken group.Along with the very fast development of Protocols in Molecular Biology and chicken genome research, breeding is more accurate, the molecular breeding faster that is in progress has climbed up the stage of history, the mechanism of many toes proterties generation is studied from molecular biology angle, and study candidate gene and molecular genetic marker, use the method for marker assisted selection can reach the object of breeding fast and efficiently.
Many toes/refer to (polydactyly) are that the common a kind of limbs of vertebrates are abnormal, and many toes phenotype of many toes phenotype of chicken and people, mouse is similar.The normal toe phenotype of chicken is the toe number of every side is 4, and the phenotype of many toes of chicken is divided into two kinds: a kind of is the increase of toe number; Another kind is toe number is 4, but the increase of toe joint number, this is a kind of specific form (Warren, 1944) of many toes trait phenotypes.Studies have found that in the growth forming process of animal foot, the abnormal expression of Shh gene and many toes have direct relation (Riddle et al., 1993; Tickle, 2003).The intron region being arranged in the LMBR1 gene of Shh upstream region of gene Mb distance specificly to regulate Shh gene at the height of the expression level of four toes.In the growth course of the mankind, mouse, cat, in LMBR1 gene intron 5, having of the sudden change of mononucleotide and the unconventionality expression of Shh gene and many toes trait phenotypes associates (Hill et al., 2003 significantly; Masuya et al., 2007).
SNP (single nucleotide polymophism), i.e. single nucleotide polymorphism causes nucleotide sequence polymorphic because single core thuja acid changes.Existing a lot of proven technique detects SNP, as single strand conformation polymorphism (PCR-SSCP), DNA sequencing, restriction fragment polymorphism (PCR-RFLP), allele specific oligonucleotide hybridization, DNA chip and TaqMan etc. at present.PCR-SSCP method is the most conventional, but existing problems have: detecting step is more; Longer (12-20 hour) consuming time; The assorted band of easy appearance, increase misjudgement probability, the confidence level of result is lower.Although DNA sequencing, allele specific oligonucleotide hybridization, DNA chip and the accuracy of TaqMan method are all higher, testing cost is done, and applies to actual breeding more difficult.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of SNP marker relevant to chicken many toes proterties and application thereof.
In order to realize the object of the invention, first the present invention provides a kind of SNP marker relevant to chicken many toes proterties, and described molecule marker is from LMBR1 gene, and its nucleotide sequence is as shown in SEQID No.1, and 246bp place is mutational site, and base is A or C.
This site is the 4645th of the 5th intron of LMBR1 gene (GenBank AccessionNumberNC_006089) simultaneously.
Further, many toes proterties is shown when the base in described mutational site is A.
Present invention also offers the primer pair for the described SNP marker that increases, in described primer pair, forward primer U as shown in SEQ ID No.2, reverse primer D is as shown in SEQ ID No.3.
Present invention also offers the test kit for detecting described SNP marker, described test kit contain forward primer U as shown in SEQ ID No.2, reverse primer D is as shown in SEQ ID No.3.
Present invention also offers a kind of method detecting chicken many toes character gene type, described method is:
With the genomic dna of chicken to be measured for template, utilize the primer pair described in claim 3, increased by PCR reaction, the pcr amplification product of gained is cut with restriction enzyme BsrDI enzyme, the digestion products that electrophoresis detection obtains, if electrophoresis showed is two band, then the genotype of chicken to be measured is CC type; If electrophoresis showed is a band, then the genotype of chicken to be measured is AA type; If electrophoresis showed is three band, then the genotype of chicken to be measured is AC type.
Further, if electrophoresis showed is two band, sequence length is respectively 144bp, 245bp, then the genotype of chicken to be measured is CC type, is wild-type homozygous body; If electrophoresis showed is a band, sequence length is 389bp, then the genotype of chicken to be measured is AA type, is mutant-type homozygotes; If electrophoresis showed is three band, sequence length is respectively 144bp, 245bp and 389bp, then the genotype of chicken to be measured is AC type, is heterozygote.
Present invention also offers a kind of test kit detecting chicken many toes character gene type, described test kit contains aforementioned primer pair and restriction endonuclease BsrDI.Described test kit also can comprise other reagent carried out needed for PCR-RFLP, and these reagent all can obtain from commercial channels.
Present invention also offers described SNP marker and determine the application in Beijing Fatty Chicken and Silky fowl many toes proterties.
The method that present invention also offers aforementioned detection chicken many toes character gene type is determining the application in Beijing Fatty Chicken and Silky fowl many toes proterties.
Further, the individual toe the Characters being CC type when genotype is 4 toes, and genotype is the individual toe the Characters of AA type and AC type is many toes.
Beneficial effect of the present invention is:
The method of detection Beijing Fatty Chicken provided by the present invention and Silky fowl many toes character gene type or test kit, the 4645th deoxynucleotide can determining the 5th intron of the LMBR1 gene (GenBankAccessionNumber NC_006089) of chicken to be measured is wild-type C or saltant type A, thus determines Beijing Fatty Chicken and Silky fowl many toes proterties: wild-type homozygous body CC shows as four toe proterties; Mutant-type homozygotes AA, heterozygote AC show as many toes proterties.
Utilize SNP marker provided by the invention, detection method or test kit, whether detection Beijing Fatty Chicken and Silky fowl carry many toes proterties mutation allele, can determine Beijing Fatty Chicken and Silky fowl chicken many toes proterties.In actual production and breeding, can select as required retain or reject many toes proterties, reject effect completely, accurately.There is provided carcass to identify as Beijing Fatty Chicken and Silky fowl high-quality chicken kind chicken with for commercial chicken many toes proterties, fundamentally protect intellecture property and the consumer's interests of breeding units, obtain considerable economic benefit.The present invention carries out marker assisted selection for many toes proterties in Beijing Fatty Chicken and Silky fowl breeding work, provide a molecular mark detection method, the method and genetic marker is used to carry out marker assisted selection to Beijing Fatty Chicken and Silky fowl, greatly can reduce molecular breeding cost, and can reject or retain Beijing Fatty Chicken and Silky fowl part chicken in actual production accurately, completely and show as non-multi toe proterties problem, for the molecular breeding accelerating to improve Beijing Fatty Chicken and Silky fowl many toes proterties is laid a good foundation.Method provided by the invention or test kit simple to operate, only digestion products need be carried out agarose gel electrophoresis, can determine that the 4645th deoxynucleotide of the 5th intron of the LMBR1 gene (GenBank AccessionNumber NC_006089) of chicken to be measured is wild-type C or saltant type A.Accuracy is high, not assorted band; Consuming time short, 4-6 hour; Low cost; Aulomatizeted Detect can be realized.For the breeding work of Beijing Fatty Chicken and Silky fowl provides convenient, play a great role in the breeding of Beijing Fatty Chicken and Silky fowl.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of PCR primer in the embodiment of the present invention 1..
Fig. 2 is the cleavage sequence figure of BsrDI in the embodiment of the present invention 1.
Fig. 3 is the agarose gel electrophoresis figure of digestion products in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
In the following example, method therefor is ordinary method if no special instructions.Raw materials usedly all can to obtain from commercial channels.Primer synthesis and examining order complete by Hua Da gene.
Embodiment 1
The foundation of the method for embodiment 1, detection Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele
One, the determination of C4645A pleomorphism site
1, pcr amplification
Beijing Fatty Chicken pure lines are selected to be experiment material.Design primer according to GenBank AccessionNumberNC_006089, the object fragment that order amplification obtains comprises the 4645th deoxynucleotide of the 5th intron of GenBankAccessionNumber NC_006089, and primer sequence is as follows:
U (upstream primer): 5 '-GCGATTTCCTCTCACCCACA-3 ' (SEQ IDNo.2)
D (downstream primer): 5 '-AGCTGAGCAACATGACAGCA-3 ' (SEQ IDNo.3)
With Beijing Fatty Chicken pure lines genomic dna for template, primer U and D is used to carry out pcr amplification.
Amplification system is as follows:
Chicken genomic dna 50ng
10X pcr amplification damping fluid 1.5 μ ι
DNTP (4mmol/L) mixed solution 0.8 μ ι
The each 0.3 μ ι of upstream and downstream primer (10pmol/L)
Taq archaeal dna polymerase 0.5U
Mg
2+ 2.5mmol/L (final concentration)
Use ddH
2o postreaction system to 15 μ ι
PCR reaction conditions is: 95 DEG C of sex change 5min; Cycling program is: 95 DEG C of sex change 20s, 68 DEG C of annealing 30s, and 72 DEG C extend 30s, totally 35 circulations; Last 72 DEG C extend 10min.
Gained pcr amplification product, carries out 1.5% agarose gel electrophoresis, and as shown in Figure 1, the clip size of pcr amplification product is 389bp to electrophorogram.
2, rflp analysis
Use restriction enzyme BsrDI, enzyme cuts the pcr amplification product in step 1.As shown in Figure 2, wherein arrow pointed location is restriction enzyme site to the cleavage sequence of BsrDI, and N represents any base.Endonuclease reaction system is as follows:
PCR reaction mixture 8 μ ι
10X enzyme cutting buffering liquid 1.6 μ ι
Restriction endonuclease BsrDI (10U/ μ ι) 1 μ ι
ddH
2O 13.4μι
Cumulative volume 24 μ ι
1-3 hour is hatched in water bath with thermostatic control 65 DEG C
Digestion products carries out 1.5% agarose (voltage 8V/cm, time 30min).Use gel imaging system is observed, and result as shown in Figure 3.
As seen from Figure 3, digestion products is 3 kinds of banding patterns.Part sample shows two electrophoretic bands, and sequence length is respectively 144bp, 245bp; Part sample is shown as an electrophoretic band, and sequence length is 389bp; Remaining sample is shown as three electrophoretic bands, and sequence length is respectively 144bp, 245bp and 389bp, and wherein length is two electrophoretic band brightness sums of 144bp, 245bp is the brightness of 389bp length electrophoretic band.
3, digestion products order-checking and sequential analysis
According to PCR-RFLP analytical results, respectively the pcr amplification product sepharose of the sample of the different electrophoretic band of display is reclaimed test kit (Tian Gen biochemical technology company limited) and reclaim purifying, the DNA fragmentation reclaimed carries out nucleotide sequencing, sequencing result shows that the expanding fragment length of different sample is 389bp, only there is the difference (C/A) of a deoxyribonucleotide, this deoxyribonucleotide is the 4645th deoxynucleotide of the 5th intron of GenBank AccessionNumber NC_006089, called after C4645A.
According to sequencing result and PCR-RFLP result, genotype is limited as follows:
If the allelotrope of this point is C, its homozygote genotype is that CC, PCR-RFLP are detected as two electrophoretic bands, and sequence length is respectively 144bp, 245bp;
If the allelotrope of this point is A, its homozygote genotype is that AA, PCR-RFLP are detected as an electrophoretic band, and sequence length is 389bp;
This site heterozygote genotype is that AT, PCR-RFLP are detected as three electrophoretic bands, and sequence length is respectively 144bp, 245bp and 389bp, and wherein length is two electrophoretic band brightness sums of 144bp, 245bp is the brightness of 389bp length electrophoretic band.
4, the preparation of Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele test kit
Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele test kit comprise: pair of primers, and the nucleotide sequence of one of them primer is sequence 2 in sequence table, and the nucleotide sequence of another primer is sequence 3 in sequence table; Restriction endonuclease BsrDI; All reagent in all reagent in step 1 in PCR reaction system and step 2 in endonuclease reaction system.
Two, the association analysis of chicken genotype and many toes proterties
Beijing Fatty Chicken pure lines are selected to be experiment material.
Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele test kit is utilized to detect.With the genomic dna of chicken to be detected for template, according to 1 carrying out pcr amplification in step one, according to 2 carrying out rflp analysis in step one.Detect that chicken is no many toes proterties, analyze the cognation of chicken genotype and many toes proterties.
Chicken genotype is as shown in table 1 with associating of many toes shape.Detected 236 chicken kinds, 181, CC type, 51, AC type, 4, AA type.The individual toe proterties of all CC types all shows as 4 toes, and all AA types and the individual toe proterties of AC type all show as many toes.
Above results proved that C4645A is polymorphic with Beijing Fatty Chicken many toes proterties close association, can as the molecule marker of Beijing Fatty Chicken many toes character screening.
Table 1LMBR1 gene C 4645A loci gene type associates with many toes proterties
Embodiment 2 detects by Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele detection kit situation and the genotype-chicken many toes trait associations check analysis that different varieties chicken carries many toes proterties mutation allele
Beijing Fatty Chicken, Silky fowl and Bai Lai is selected to navigate laying hen as experiment material.
Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele detection kit is utilized to detect the C4645A of above-mentioned different varieties chicken polymorphic.Beijing Fatty Chicken and Silky fowl many toes proterties mutation allele detection kit comprise: pair of primers, and the nucleotide sequence of one of them primer is sequence 2 in sequence table, and the nucleotide sequence of another primer is sequence 3 in sequence table; Restriction endonuclease BsrDI; This test kit also comprises the conventional reagent needed for PCR-RFLP.With the genomic dna of chicken to be detected for template, carry out pcr amplification according to 1 of step one in embodiment 1, carry out rflp analysis according to step 2 in embodiment 1.
Analyze three kind chicken C4645A allelotrope and genotypic distribution situation.Three kind chicken C4645A allelotrope and genotypic distribution as shown in table 2.
Table 2LMBR1 gene C 4645A allelotrope and the distribution of genotype in different chicken
From table 2, carry in Beijing Fatty Chicken that allele C frequency is 0.875, to carry mutation allele A frequency be 0.125, the genotypic frequency of CC is 0.767, the genotypic frequency of AC is 0.216, the genotypic frequency of AA is 0.017, the result that chicken toe proterties detects is shown that all AA, AC genotype individuals are many toes proterties, all CC genotype individuals are four toe proterties, this is consistent with phenotypic evaluation result, also studies with other and reports consistent (Meng He etc. 2009).Carry in Silky fowl that allele C frequency is 0.237, to carry mutation allele A frequency be 0.763, the genotypic frequency of CC is 0.082, the genotypic frequency of AC is 0.311, the genotypic frequency of AA is 0.607, the result that chicken toe proterties detects is shown that all AA, AC genotype individuals are many toes proterties, all CC genotype individuals are four toe proterties, and this is consistent with phenotypic evaluation result.Bai Laihang laying hen does not carry mutation allele A, does not have AA genotype and AC genotype, and the result of chicken being carried out to the detection of many toes proterties shows that this is consistent with the fact not containing many toes phenotype in this chicken kind toe type, shows that detected result is true, reliable.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a SNP marker relevant to chicken many toes proterties, is characterized in that, described molecule marker is from LMBR1 gene, and its nucleotide sequence is as shown in SEQ ID No.1, and 246bp place is mutational site, and base is A or C.
2. SNP marker according to claim 1, is characterized in that, shows many toes proterties when the base in described mutational site is A.
3., for the primer pair of the SNP marker described in claim 1 or 2 that increases, it is characterized in that, in described primer pair, forward primer U as shown in SEQ ID No.2, reverse primer D is as shown in SEQ ID No.3.
4. require the test kit of SNP marker described in 1 or 2 for test right, it is characterized in that, described test kit contain forward primer U as shown in SEQ ID No.2, reverse primer D is as shown in SEQ ID No.3.
5. detect a method for chicken many toes character gene type, it is characterized in that, described method is:
With the genomic dna of chicken to be measured for template, utilize the primer pair described in claim 3, increased by PCR reaction, the pcr amplification product of gained is cut with restriction enzyme BsrDI enzyme, the digestion products that electrophoresis detection obtains, if electrophoresis showed is two band, then the genotype of chicken to be measured is CC type; If electrophoresis showed is a band, then the genotype of chicken to be measured is AA type; If electrophoresis showed is three band, then the genotype of chicken to be measured is AC type.
6. method according to claim 5, is characterized in that, if electrophoresis showed is two band, sequence length is respectively 144bp, 245bp, then the genotype of chicken to be measured is CC type, is wild-type homozygous body; If electrophoresis showed is a band, sequence length is 389bp, then the genotype of chicken to be measured is AA type, is mutant-type homozygotes; If electrophoresis showed is three band, sequence length is respectively 144bp, 245bp and 389bp, then the genotype of chicken to be measured is AC type, is heterozygote.
7. detect a test kit for chicken many toes character gene type, it is characterized in that, described test kit contains primer pair described in claim 3 and restriction endonuclease BsrDI.
8. the SNP marker described in claim 1 or 2 is determining the application in Beijing Fatty Chicken and Silky fowl many toes proterties.
9. the method described in claim 5 or 6 is determining the application in Beijing Fatty Chicken and Silky fowl many toes proterties.
10. application according to claim 9, is characterized in that, genotype is the individual toe the Characters of CC type is 4 toes, and genotype is the individual toe the Characters of AA type and AC type is many toes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506086A (en) * | 2015-12-25 | 2016-04-20 | 华中农业大学 | SNP molecular markers related to chicken-fertilization duration time characters and application thereof |
| CN106244713A (en) * | 2016-09-22 | 2016-12-21 | 北京市农林科学院 | A kind of method detecting Beijing Fatty Chicken five toe character and application |
| CN113151509A (en) * | 2021-06-04 | 2021-07-23 | 河南农业大学 | Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application |
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| CN105506086A (en) * | 2015-12-25 | 2016-04-20 | 华中农业大学 | SNP molecular markers related to chicken-fertilization duration time characters and application thereof |
| CN105506086B (en) * | 2015-12-25 | 2019-06-21 | 华中农业大学 | The relevant SNP marker of chicken fertilization duration character and its application |
| CN106244713A (en) * | 2016-09-22 | 2016-12-21 | 北京市农林科学院 | A kind of method detecting Beijing Fatty Chicken five toe character and application |
| CN106244713B (en) * | 2016-09-22 | 2020-02-21 | 北京市农林科学院 | Method for detecting five-toe characters of Beijing fatty chicken and application thereof |
| CN113151509A (en) * | 2021-06-04 | 2021-07-23 | 河南农业大学 | Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application |
| CN113151509B (en) * | 2021-06-04 | 2023-09-05 | 河南农业大学 | Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application |
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