CN101967480B - Molecular marker relevant to chicken skin color and authentication method and application thereof - Google Patents
Molecular marker relevant to chicken skin color and authentication method and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker relevant to chicken skin color, which is an A/G basic group mutation located in the 259th basic group in the sequence disclosed in SEQ ID NO:1. The invention also provides an authentication method of the molecular marker and an application thereof in molecular marker assisted selection on the aspect of chicken skin color characters. The molecular marker takes the DNA sequence of a chicken BCDO2 gene and selects primers shown in SEQ ID NO:2 and SEQ ID NO:3 for amplification. The detection method used by the invention has high accuracy and low cost. The detection of the A/G mutant site can know the gene type of the character of the target chicken flock skin color so as to select chicken flocks on purpose and improve the consistency between chicken flock skin color and shank color.
Description
Technical field
The present invention relates to chicken marker assisted selection technical field, specifically, relate to a kind of for the cock skin skin color of chicken marker assisted selection relevant molecule marker and discrimination method and application.
Background technology
Skin color is for one of important indicator of estimating bird product economy value, the yellow of cock skin skin is mainly the result due to yellow pigmentations such as carotenoid and xenthophylls, and this deposition capability is subjected to the impact of the aspects such as heredity, feed nutrition, fodder additives, feeding environment, healthy state.The performance of cock skin skin epidermal area Lutein is mainly by W
+, this locus of w determines, this gene is positioned on number one karyomit(e), belongs to the 3rd linkage group.The dominant gene of wild-type can hinder the deposition of yellow pigment on the positions such as skin, pin shin, beak and fat, makes skin, pin shin color present white; The recessive gene of relative mutant allows yellow pigment in position depositions such as skin, pin shin, beak and fat, makes these positions present yellow.Research is at present found, the major function of BCDO2 (the beta-carotene dioxygenase 2) gene of coding β-carotene dioxygenase 2 is that coloured carotenoid is decomposed into colourless material, thereby make the cock skin skin there is no yellow pigmentation, and become pale skin.Studying also proves, and the sudden change on this gene is relevant with cock skin skin color, but at present from the angle of heredity, also not setting up a kind of effective differentiation chicken is yellow skin or pale skin, and pale skin is heterozygote or homozygous method.
Summary of the invention
One of purpose of the present invention is to provide the relevant molecule marker of a kind of cock skin skin color.This molecule marker is positioned on the exon of cock skin skin Color Related Gene BCDO2.Specifically, this molecule marker is arranged in the 259th bit base A/G of place base mutation of sequence shown in SEQ ID NO:1.When this place's base was A, cock skin skin color was white, and when this place's base was G, cock skin skin color was yellow.
The present invention take with the DNA full length sequence of cock skin skin Color Related Gene BCDO2 as the basis, seek the mutational site and the corresponding pleiomorphism detecting method that affect skin color on this gene, by the checking in a plurality of colonies, for chicken breeding provides a kind of molecular identification method about its skin color.Below concrete technical scheme:
Take the DNA sequence dna (reference sequences GeneBank accession number is as NC 006111.2) of chicken BCDO2 gene as template, this Gene Partial sequence of design primer amplification, primer used is:
Forward primer: 5 '-TCCTCTGATTGCTTTACTGACTTG-3 ',
Reverse primer: 5 '-GGGAAGGAGGTATCATTGGAGA-3 '.
The sequence that obtains of increasing comprise the 6th complete exon and part the 5th intron, part the 6th intron, as described in sequence table SEQ ID NO:1, this sequence total length is 444bp.
The Multiple Sequence Alignment analysis of the present invention by the above-mentioned DNA fragmentation that obtains being carried out (comprise pale skin chicken and yellow skin chicken) between chicken breed, the sequence of acquisition as shown in Figure 2.Comparison result is found a kind of molecule marker that the chicken marker assisted selection is used that can be used as, and this mark is relevant with the skin color of chicken, exists A/G to suddenly change at the 259th bit base place of this sequence, causes Sdu I-RFLP polymorphism.
Second purpose of the present invention has been to provide the discrimination method of this molecule marker, take the DNA sequence dna of chicken BCDO2 gene as template, selects following primer to increase:
Forward primer: 5 '-TCCTCTGATTGCTTTACTGACTTG-3 ',
Reverse primer: 5 '-GGGAAGGAGGTATCATTGGAGA-3 ';
The resulting product utilization Sdu of pcr amplification I restriction endonuclease is carried out endonuclease reaction, agarose gel electrophoresis detects enzyme and cuts product, if only have the band of a 444bp, the DNA double chain is the A base at this place, mutational site, be judged as the AA genotype, show as pale skin; If 2 bands that are respectively 263bp and 181bp are arranged, illustrate that the DNA double chain is the G base at this place, mutational site, be judged as the GG genotype, show as yellow skin; If 3 bands that are respectively 444bp, 263bp and 181bp are arranged, illustrate that the DNA double chain is the A base at this chain in place, mutational site, another chain is the G base, is judged as the AG genotype, because A allelotrope is dominant, shows as pale skin.
The 3rd purpose of the present invention is to provide the application of this molecule marker in the chicken molecular marker assisted selection, and particularly cock skin skin color is selected the application in breeding.
By technical scheme provided by the present invention, can also be prepared into the identification reagent box of this molecule marker.
The invention has the beneficial effects as follows, by the detection to above-mentioned A/G mutational site, can know the genotype of this proterties of target chicken group skin color, thereby can have purpose that the chicken group is selected, improve the consistence of its offspring chicken group's skin and shin look.In addition, detection method accuracy used in the present invention is high, cost is low, efficient is high.
Description of drawings
Fig. 1 is the partial dna sequence on chicken BCDO2 gene in embodiment 1, is used for seeking the electrophoresis picture of the fragment of molecule marker;
Fig. 2 uses the result that method of the present invention is compared to this fragment on the BCDO2 gene of 2 pale skin chickens and 2 yellow skin chickens in embodiment 1;
Fig. 3 is three kinds of genotype (AA, AG and the GG) electrophoretograms (agarose gel concentration is 1.5%) of the Sdu I-RFLP on chicken BCDO2 gene extron 6 in embodiment 1.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
Embodiment 1: the foundation of cock skin skin color molecular detecting method
(1) primer:
Take the DNA sequence dna (reference sequences GeneBank accession number is as NC 006111.2) of chicken BCDO2 gene as template, design pair of primers YSD-F/YSD-R clone obtains the DNA sequence dna (comprising the 6th complete exon and part the 5th intron, part the 6th intron) as shown in sequence SEQ ID NO:1, and this sequence total length is 444bp.Primer sequence used is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3, and is specific as follows:
YSD-F：5′-TCCTCTGATTGCTTTACTGACTTG-3′，
YSD-R：5′-GGGAAGGAGGTATCATTGGAGA-3′。
(2) pcr amplification condition:
Cumulative volume is to add DNA profiling 1 μ L in the PCR reaction system of 25 μ L, each 1 μ L before and after 2 * PCR reaction mixture, 12.5 μ L, 10mM primer, and Taq archaeal dna polymerase 0.625U, distilled water 8.25 μ L, the PCR reaction conditions is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 33 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
(3) seek molecule marker:
Choose the blood DNA of 2 pale skin chickens and 2 yellow skin chickens and do template, carry out pcr amplification, the PCR product detects through 1.5% agarose gel electrophoresis, as shown in Figure 1.Fig. 1 is the partial dna sequence on chicken BCDO2 gene, be used for seeking the electrophoresis picture of the fragment of molecule marker, clip size is 444bp (agarose gel concentration is 1.5%), wherein, the M swimming lane is DNA molecular amount standard (DL2000), B1, B2 represent pale skin chicken BCDO2 gene PCR amplified fragments, and H1, H2 represent yellow skin chicken BCDO2 gene PCR amplified fragments.The PCR product is reclaimed, clones, checks order, the nucleotide sequence of its sequencing sequence as shown in H1, H2, B1, B2 in Fig. 2.Find to exist 8 SNP of place by Cluster W comparison, the deletion mutantion of 1 place, compare with the pale skin chicken and find that the yellow skin chicken at 259 places, base mutation has occured, sport G by A, the position of this mutational site on BCDO2 genome sequence (the GeneBank accession number is NC 006111.2) is 14253 places.Specifically see Fig. 2, Fig. 2 is the result that this fragment on the BCDO2 gene of 2 pale skin chickens and 2 yellow skin chickens is compared.In figure, B1, B2 represent the pale skin chicken, and H1, H2 represent the yellow skin chicken, the A/G mutational site that to be this sequence exist at 259 bit base places in figure shown in square frame, and the sequence shown in underscore is the primer sequence that the present invention designs.The experiment concrete operation step is as follows:
1, the purifying of PCR product: downcut from the low melting-point agarose gel gel that contains the purpose fragment under ultraviolet lamp, put into 1.5mL Ependorff pipe, then use PCR product purification test kit (available from Promega company) purified pcr product, according to this test kit specification sheets operation.
2, ligation: PCR product and the pMD18-T Vector (available from TaKaRa company) of purifying are connected, and the ligation cumulative volume is 10 μ L, connects damping fluid, 2.5 μ L comprising 2 * fast; Carrier (50ng), 0.5 μ L; Reclaim DNA, 7-8 μ L.Bullet is beaten and is mixed a little, in 16 ℃ connect 1h more than or 4 ℃ of connections spend the night.
3, the preparation of competent cell: cultivated on the fresh flat board of 16-20h the single colony inoculation of DH5 α of picking from 37 ℃ of incubators in 2mL LB, in 37 ℃ of shaking table shaking culture 3h, switching 1mL bacterium liquid is in the saline bottle that contains 300mL LB, continuation is at the about 4h of 37 ℃ of shaking table shaking culture, when OD600 reaches 0.3-0.4, saline bottle is taken out from shaking table and put the cooling 10-15min of ice bath, then bacterium liquid is changed in centrifuge tube in 4 ℃ 4, the centrifugal 10min of 000g is with collecting cell, centrifuge tube is inverted to abandon clean nutrient solution, ices the CaCl of the 0.1mol/L of precooling with 10mL
2Resuspended precipitation, ice bath 30min repeats 4 ℃ 4, and the centrifugal 10min of 000g once ices the CaCl of the 0.1mol/L of precooling with 4ml
2Resuspended precipitation is put 4 ℃ and is saved backup.
4, transform: add 100 μ L competent cells in the 1.5mL centrifuge tube of sterilization, add on ice to connect product 5 μ L, with liquid-transfering gun piping and druming evenly, ice bath 30min; Centrifuge tube is placed in the circulator bath (not vibrations) of 42 ℃, after heat shock 90s, rapid ice bath 2min; Add again 400 μ L LB nutrient solutions in centrifuge tube, bathe recovery 45-60min in 37 ℃ of shaking tables (200rpm/min) temperature; Centrifugal, remove the part supernatant liquor, the bacterium liquid of getting after 100 μ L recover is distributed on the flat board that contains Amp, smoothens; After liquid fully absorbs, be inverted plate, in 37 ℃ of cultivations 14-16 hour, observation had or not bacterium colony to grow.
5, the evaluation of positive colony and order-checking: the bacterial plaque that picking transforms from flat board accesses the 1.5mL centrifuge tube that contains 400 μ L LB and in 37 ℃ of shaking table joltings and cultivates approximately 8h left and right.Take this bacterium liquid as template, select the universal primer M13 (Beijing Liuhe Huada Genomics Technology Co., Ltd provides) of former primer or order-checking to carry out pcr amplification (annealing temperature 58-60 ℃).Electrophoresis detection, the bacterium liquid of picking positive colony is sent to order-checking, and sequencing is completed by upper Beijing Liuhe Huada Genomics Technology Co., Ltd.
In the square example of this enforcement, pcr amplification product is special PCR product (Fig. 1) through 1.5% agarose gel electrophoresis detected result demonstration, Fig. 1 is the partial dna sequence on chicken BCDO2 gene in embodiment 1, be used for seeking the electrophoresis picture of the fragment of molecule marker, clip size is 444bp (agarose gel concentration is 1.5%), wherein, the M swimming lane is DNA molecular amount standard (DL2000).With the recovery of PCR product, purifying rear clone, order-checking (comparison chart of measured sequence is seen Fig. 2), Fig. 2 uses the result that method of the present invention is compared to this fragment on the BCDO2 gene of 2 pale skin chickens and 2 yellow skin chickens, wherein B1, B2 represent pale skin chicken aligned sequences, H1, H2 represent yellow skin chicken aligned sequences, and square frame is depicted as this sequence in the A/G mutational site of 259 bit base place's existence.Result shows that resulting DNA sequence dna length is 444bp, comprises the 6th complete exon, and part the 5th intron and part the 6th intron sequences (as shown in sequence table SEQ ID NO:1); Sequencing result shows at 259 places of this DNA sequence dna and has the A/G base mutation.
(4) the PCR-RFLP detection method is set up:
The A/G base mutation at this fragment 259 places, just can be identified by restriction endonuclease Sdu I, with above-mentioned PCR product 6 μ L, 10 * damping fluid, 1 μ L, restriction enzyme Sdu I 0.2 μ L (2U), adding distilled water mends to 10 μ L, with centrifugal after sample blending, 37 ℃ of incubators are placed 6h, detect with 1.5% agarose gel electrophoresis, imaging system is observed enzyme and is cut result, records genotype.This gene mutation site is controlled by two allelotrope, and wherein A is the allelotrope that does not form restriction enzyme site, and G is the allelotrope that forms restriction enzyme site.These two allelotrope can form three kinds of genotype, wherein the AA type is that homozygous (the only having DNA band of 444bp during electrophoresis detection) that enzyme is cut do not occur, homozygous (the occurring two DNA bands of 263bp and 181bp during electrophoresis detection) that the GG type is cut for enzyme occurs, AG is heterozygous (occurring three DNA bands of 444bp, 263bp and 181bp during electrophoresis detection), sees Fig. 3 for details.Fig. 3 is three kinds of genotype (AA, AG and the GG) electrophoretograms (agarose gel concentration is 1.5%) of the Sdu I-RFLP on chicken BCDO2 gene extron 6 in the present invention, and the M swimming lane is DNA molecular amount standard (DL2000).
Detection and the application of embodiment 2:PCR-Sdu I-RFLP polymorphism distribution situation in each chicken strain
Having collected different shin looks and the skin-color chicken blood DNA of totally 3 strains in this example, is respectively B, Z, H totally 3 strains, and sample standard deviation is from Guangdong intelligence prestige agricultural science and technology company limited by shares.According to the described method of embodiment 1, above chicken kind has been carried out PCR-Sdu I-RFLP and detected, to verify that this kind of the present invention is about the molecular diagnosis method of cock skin skin color.Detected result is as shown in table 1.Table 1 is the distribution detected result of BCDO2 gene Sdu I-RFLP polymorphism in the chicken group.
Table 1
As shown in Table 1, the genotype of the individuality that detects all conforms to its skin color, illustrates that method of the present invention is reliable, effective.
Molecule marker of the present invention can be applicable to cock skin skin color and selects in breeding.The Z Strains of Chickens that detects as table 1, its macroscopic features is that skin color is white, the shin look is black, and find that in our testing process in its colony, some pale skin individualities are AG heterozygous genes type, if the stud mating of AG heterozygous genes type so, the GG genotype will occur in its offspring colony, show as yellow individuality, and yellow skin is the macroscopic features that does not meet this strain of Z.The individuality of this strain father, female Dai Zhongwei AG heterozygous genes type can be weeded out so use molecule marker of the present invention, to guarantee not have in its offspring chicken group the appearance of yellow skin individuality, make colony consistent aspect this macroscopic features of skin color.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Sequence table
＜120〉relevant molecule marker and discrimination method and the application of a kind of cock skin skin color
<160>3
<170>PatentIn version 3.2
<210>1
<211>444
<212>DNA
<213>chicken
<222>(1)...(444)
<400>1
tcctctgatt gctttactga cttgataact gtttctttat tctttttttt tctgcccagt 60
tatgtaacta ctgtatttct acctacacta aggaaataga aaagcactga gcctgacttt 120
atctttgtga atctgggcag cttcacatct cagtggtgct gaaccccctt ttttctttcc 180
agggactaca tataatataa ttgaggtccc tccacaaaag tcgaactgca atgagacctt 240
agagggagca aaagtgctat gctccattgc tccaacagat aacatgaaac cctcctacta 300
tcacagcttt ggtaagaatg cttcctgtct catactccat ggatttagcc tcctgtgcag 360
cacaaggggc tgttaactgc cctctgtttc aatttttttt gttgttgttt catttgccaa 420
gttctccaat gatacctcct tccc 444
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<222>(1)...(24)
<400>2
tcctctgatt gctttactga cttg 24
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<222>(1)...(22)
<400>3
gggaaggagg tatcattgga ga 22
Claims (4)
1. a molecular detecting method of differentiating cock skin skin color, is characterized in that, as molecule marker, detects the base A/G of the place base mutation of described the 259th with the base of the 259th in sequence as shown in SEQ ID NO:1.
2. molecular detecting method according to claim 1, is characterized in that, comprises the following steps: take the DNA sequence dna of the coding β-carotene dioxygenase 2 gene BCDO2s relevant with cock skin skin color as template, select following primer amplification:
Forward: 5 '-TCCTCTGATTGCTTTACTGACTTG-3 ',
Oppositely: 5 '-GGGAAGGAGGTATCATTGGAGA-3 ';
The gained amplified production is cut with restriction enzyme Sdu I enzyme,
If only have a band, this place, mutational site is the A base;
If two bands are arranged, the place, mutational site is the G base;
If three bands are arranged, illustrate that this chain in place, mutational site is the A base, another chain is the G base.
3. the application in the molecular marker assisted selection of method claimed in claim 1 aspect cock skin skin color proterties.
4. application according to claim 3, is characterized in that, comprises the application in cock skin skin color selection breeding.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103834642B (en) * | 2014-02-14 | 2016-06-29 | 华南农业大学 | Molecular marker that a kind of chicken skin color is relevant and application thereof |
| CN105603094B (en) * | 2016-02-22 | 2019-01-04 | 杭州市农业科学研究院 | A kind of relevant SNP marker of black-bone chicken colour of skin character and application |
| CN110157814B (en) * | 2019-05-31 | 2022-05-10 | 江苏省家禽科学研究所 | SNP molecular marker related to drumstick skin hair follicle density character and detection method and application thereof |
| CN111876492B (en) * | 2020-07-15 | 2021-07-09 | 华南农业大学 | Molecular marker influencing skin color of shank of chicken and application thereof |
| CN112899374B (en) * | 2021-03-04 | 2022-06-07 | 华南农业大学 | Molecular marker related to skin color of chicken, primer group and application |
| CN113218884B (en) * | 2021-04-29 | 2022-10-14 | 华南农业大学 | Breeding method for chicken skin yellowness |
| CN115521984A (en) * | 2021-06-25 | 2022-12-27 | 华中农业大学 | Method for identifying nondestructive sexes in middle stage of chicken embryo by utilizing tibia heredity and transmission spectrum |
| CN115181805B (en) * | 2022-02-23 | 2023-08-11 | 华南农业大学 | Molecular marker related to yellow-feather broiler leg skin yellowness and application thereof |
| CN115992264A (en) * | 2023-02-21 | 2023-04-21 | 华中农业大学 | Chicken yellow and white skin color molecular identification primer group and method thereof |
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| WO2009046724A2 (en) * | 2007-10-08 | 2009-04-16 | Aarhus Universitet | Polymorphisms of mbl |
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