CN111394473B - Molecular marker related to chicken antler crowns and typing method and application thereof - Google Patents
Molecular marker related to chicken antler crowns and typing method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a molecular marker related to chicken antler crowns, and a typing method and application thereof. The molecular marker typing method related to the antler corona is characterized in that multiple PCR amplification is carried out on a sample blood DNA sample after the sample blood DNA sample is extracted, an extension reaction is carried out after a PCR product is purified, the product is sequenced to type a variation site, and then heterozygote individuals are eliminated. The invention can be used for purifying the 'antler' crown character in breeding and for cultivating a new strain with the 'antler' crown character and a new mating line.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a molecular marker related to chicken antler crowns, and a typing method and application thereof.
Background
The appearance characteristic is usually the symbolic character of the variety, so the appearance breeding is particularly important in chicken breeding and breed conservation, and the appearance characteristic is also an important basis for common consumers to distinguish the variety, particularly the local variety. The shape of the cockscomb is one of the mark characteristics of some varieties, just like the rose cockscomb is an important characteristic of Taihe and Yu black-bone chickens in China, the hair cockscomb is an important characteristic of Beijing oil chickens, the double cockscomb is an important appearance characteristic of Imperial concubine chickens, and the antler cockscomb (the tail of the cockscomb is forked, the part of the cockscomb is provided with crown teeth, and the shape of the antler is similar to that of deer horn) is a main characteristic of the river chickens and the cave chickens in China. Antler crowns exhibited autosomal dominant inheritance relative to single crowns.
In the breeding production of animals, three schemes are provided for the selection and purification of monogenic traits: phenotype direct panning, gradually reducing the frequency of related recessive alleles in a population; and eliminating recessive allele by test cross. And thirdly, molecular marker assisted selection, and corresponding allele is directly removed. In the scheme, multiple generations are needed for direct selection and panning through phenotypes, recessive alleles always exist in a population, and the recessive alleles cannot be completely removed. The scheme 'II' can theoretically eliminate the related recessive allele through a generation, but errors are easy to occur in the actual operation process due to complicated process. The scheme III has the characteristics of rapidness and simple process, and the premise is that molecular markers which are completely linked with the characters or causative mutations related to the characters are known.
The antler crown character is a character controlled by a single gene and is dominant to the wild single crown character. At present, the mode of rejecting wild alleles in a river-field chicken/elephant-cave chicken group or a group with the 'antler' crown character synthesized by taking river-field chickens/elephant-cave chickens as materials can only adopt a scheme 'I' or a scheme 'II'. In the early research, we identify the mutation site which is completely linked with the antler, and based on the early research result, the invention provides a molecular marker and an auxiliary selection method thereof to accurately select the antler crown genotype.
Disclosure of Invention
The invention aims to provide a molecular marker-assisted selection method for detecting the genotype of an individual with the character of the chicken 'antler' crown, so as to accurately eliminate the individual carrying the wild type (single crown) allele.
The invention realizes the technical effects through the following technical scheme:
a molecular marker related to the 'antler' crown of chicken is provided with a gene sequence shown in SEQ.4 and/or SEQ.5.
A typing method of molecular markers related to the 'antler' crown of a chicken is characterized in that a sample blood DNA sample is extracted and then subjected to multiplex PCR amplification, a PCR product is purified and then subjected to extension reaction, the product is sequenced to type a variation site, and then heterozygote individuals are eliminated, wherein the variation site is the 44 th nucleotide of an amplification fragment, and the wild type allele sequence is shown as SEQ.No. 4; the sequence of the mutant gene is shown in SEQ.No. 5.
Preferably, the molecular marker related to the chicken 'antler' corona and the typing method thereof specifically comprise the following steps:
step 1: extracting sample DNA: extracting DNA from sample blood by phenol-chloroform extraction method;
step 2: typing of target sites:
(1) multiplex PCR amplification: mixing the forward primer and the reverse primer to obtain a mixed primer; adding sample blood DNA and nucleotide, adding water to constant volume to prepare a PCR system, and keeping the temperature at 95 ℃ for 3 min; 15s at 94 ℃ and 15s at 55 ℃ for 35 cycles; 30s at 72 ℃; carrying out amplification reaction under the amplification condition of 72 ℃ for 3min to obtain a PCR product;
(2) and (3) PCR product purification: the PCR product was purified using ExoI, FastAP and ExoI buffer;
(3) and (3) extension reaction: adding the purified PCR product into Snapshot Mix reagent and extension primer, and fixing the volume with water at 96 ℃ for 1 min; 10s at 96 ℃ and 5s at 52 ℃ for 30 cycles; carrying out extension reaction under the amplification condition of 30s at 60 ℃ to obtain an extension product;
(4) sequencing and typing: and (4) denaturing the extension product, sequencing the extension product by a sequencer, and eliminating heterozygote individuals according to the nucleotide of the target site.
More preferably, the molecular marking method related to the chicken 'antler' corona specifically comprises the following steps:
step 1: extracting sample DNA: DNA was extracted from the sample blood by phenol-chloroform extraction.
The extraction method of genomic DNA adopts phenol-chloroform extraction method (Ospol F et al, 1998), and comprises the following steps:
(1) putting 30 mu L of whole blood into a 1.5mL centrifuge tube, respectively adding 470 mu L of 1 xSET buffer solution, 12.5 mu L of 20% SDS and 6 mu L of 10mg/mL proteinase K, uniformly mixing, and putting in a 55 ℃ water bath for overnight;
(2) taking out a sample, putting the sample into a 1.5mL centrifuge tube, adding 500 mu L of saturated phenol, slightly shaking for 20min, and centrifuging at 10000rpm for 10 min;
(3) taking the supernatant, adding 500 μ L saturated phenol again, shaking gently for 20min, centrifuging at 10000rpm for 10 min;
(4) collecting supernatant, adding 500 μ L chloroform-isoamyl alcohol (wherein the volume ratio of chloroform to isoamyl alcohol is 23: 1), shaking for 20min, centrifuging at 10000rpm for 10 min;
(5) taking the supernatant, adding 1mL of ice absolute ethyl alcohol (-20 ℃), swinging back and forth to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out the ethyl alcohol;
(6) washing DNA once with 1mL of 75% ethanol, pouring off the ethanol, and drying in a drying oven at 50 ℃; (7) after the DNA is completely dried, 300 mu L of sterilized double distilled water is added for dissolving, and the DNA is dissolved in a water bath kettle at 50 ℃ overnight;
(8) storing in a refrigerator at-20 deg.C for use.
Step 2: typing of the site of interest
(1) Multiplex PCR amplification
Dissolving the forward primer and the reverse primer by using 1 × TE respectively until the concentration is 10pmol, uniformly mixing the forward primer and the reverse primer, and centrifuging to obtain a mixed primer; preparing a PCR system according to the following volume ratio: DNA 1-2. mu.L, 2. mu.L PCR mix 7.5. mu.L, mix primers 2. mu.L, add H2Supplementing O to 15 mu L, transferring the PCR system to a 96 micro-porous plate for PCR reaction, and carrying out 3min at 95 ℃; 15s at 94 ℃ and 15s at 55 ℃ for 35 cycles; 30s at 72 ℃; carrying out amplification reaction under the amplification condition of 72 ℃ for 3 min; wherein the sequence of the forward primer is shown as SEQ.No.1, and the sequence of the reverse primer is shown as SEQ.No. 2;
(2) PCR product purification
mu.L of the PCR product was taken, and ExoI 0.2. mu.L, FastAP 0.8. mu.L and ExoI buffer 0.7. mu.L were added thereto using H2Adding O to 7 mu L, mixing uniformly, and purifying the PCR product according to the purification conditions of 37 ℃ for 15min and 80 ℃ for 15min in sequence;
in this step, the remaining primers in the reaction product were removed by ExoI, and the remaining DNTP in the reaction was removed by FastAP.
(3) Extension reaction
The purified PCR product was subjected to an extension reaction, and an extension reaction product was prepared from the following components in the following volume ratiosComprises the following steps: after purification, 2. mu.L of PCR product, 1. mu.L of Snapshot Mix reagent, 1. mu.L of extension primer, and addition of H2Supplementing O to 6 mu L, transferring the obtained product to a 96 micro-porous plate for PCR reaction, and carrying out 1min at 96 ℃; 10s at 96 ℃ and 5s at 52 ℃ for 30 cycles; carrying out amplification reaction at the amplification condition of 30s at 60 ℃ to obtain an extension product, wherein the sequence of the extension primer is shown as SEQ.No. 3;
(4) sequencing typing
Taking 1 mu L of extension product, adding 10 mu L of sample Hidi, denaturing at 95 ℃ for 3min, immediately performing ice water bath, sequencing by a sequencer, and determining the genotype of the sample according to the sequencing result; individuals with the genotype of GG are selected and retained, and individuals with the genotype of the target site of AG are eliminated.
The wild type allele sequence of the target gene is shown as SEQ.No. 4; the mutant allele sequence of the target gene is shown as SEQ.No. 5. The difference between the two is that the 44 th nucleotide of the gene is different.
The invention also provides an application of the molecular marker, namely the application of the molecular marker in the 'antler' crown selection and elutriation.
Compared with the prior art, the invention has the beneficial technical effects that: is a direct selection aiming at the site which is completely linked with the 'antler' crown character, and is more accurate than the traditional phenotype selection. The property of the 'antler' crown after selection can not be separated, namely, a single crown individual can not appear any more.
Drawings
FIG. 1 shows the result of genotyping of a sample whose target site is AG.
FIG. 2 is a result of genotyping of a sample having GG as a target site
Detailed Description
The invention is further described below by means of specific examples, which, however, should be understood by a person skilled in the art, do not in any way limit the scope of the patent protection of the invention.
Example 1:
and (3) eliminating heterozygote individuals in the river-field chicken population so as to ensure that the offspring do not have single-crown individuals.
1. Sample collection
About 1mL of blood was drawn from the inferior vein of the chicken wings using a disposable syringe, injected into a 1.5mL centrifuge tube autoclaved and filled with about 200. mu.L of 2% sterile EDTA anticoagulant, shaken gently, the wing number recorded, and stored at-20 ℃ for later use.
DNA extraction
The extraction of genomic DNA was performed by phenol-chloroform extraction (Ospor F et al, 1998) using the following steps:
(1) putting 30 mu L of whole blood into a 1.5mL centrifuge tube, respectively adding 470 mu L of 1 xSET buffer solution, 12.5 mu L of 20% SDS and 6 mu L of 10mg/mL proteinase K, uniformly mixing, and putting in a 55 ℃ water bath for overnight;
(2) taking out a sample, putting the sample into a 1.5mL centrifuge tube, adding 500 mu L of saturated phenol, slightly shaking for 20min, and centrifuging at 10000rpm for 10 min;
(3) taking the supernatant, adding 500 μ L saturated phenol again, shaking gently for 20min, centrifuging at 10000rpm for 10 min;
(4) collecting supernatant, adding 500 μ L chloroform-isoamyl alcohol (volume ratio of the two is 23: 1), shaking for 20min, centrifuging at 10000rpm for 10 min;
(5) taking the supernatant, adding 1mL of ice absolute ethyl alcohol (-20 ℃), swinging back and forth to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out the ethyl alcohol;
(6) washing DNA once with 1mL of 75% ethanol, pouring off the ethanol, and drying in a drying oven at 50 ℃; (7) after the DNA is completely dried, 300 mu L of sterilized double distilled water is added for dissolving, and the DNA is dissolved in a water bath kettle at 50 ℃ overnight;
(8) storing in a refrigerator at-20 deg.C for use.
3. Typing of the site of interest
(1) Multiplex PCR amplification
The synthesized primers were dissolved in 1 × TE to a concentration of 10pmol, and the primers in one set were added together, mixed well and centrifuged.
Preparing a PCR system:
| DNA | 1-2μL |
| 2*PCR mix | 7.5μL |
| mixed primer | 2μL |
| H2O | Make up to 15 mu L |
Subpackaging the prepared PCR manifold into 96-well PCR plates, centrifuging, adding 2 mu LDNA sample into each well, centrifuging and loading into a PCR instrument
Specific amplification conditions were as follows:
(2) PCR product purification
After PCR amplification, 3. mu.L of PCR product was purified with ExoI and FastAP, mainly using ExoI to remove the remaining primers in the reaction product, and using FastAP to remove the remaining DNTP in the reaction
| PCR product | 3μL |
| ExoI | 0.2μL |
| FastAP | 0.8μL |
| ExoI buffer | 0.7μL |
| H2O | Make up to 7 mu L |
Carrying out extension reaction after purification at 37 ℃ for 15min and 80 ℃ for 15min, and mixing extension primers in advance.
(3) Extension reaction
The system is as follows:
| PCR product | 2μL |
| Snapshot Mix reagent | 1μL |
| Extension primer | 1μL |
| Water is supplemented to | 6μL |
The amplification conditions for the extension reaction were:
(4) sequencing typing
mu.L of the extension product was taken, 10. mu.L of Hidi sample was added, denatured at 95 ℃ for 3min, immediately subjected to ice water bath, and loaded onto a sequencer. The typing results were the following two types: the sequencing results are shown in FIG. 1 and FIG. 2, respectively. Wherein FIG. 1 shows the result of genotyping of a sample whose target site is AG. FIG. 2 shows the result of genotyping a sample whose target site is GG.
Selecting and reserving breeding hens: and selecting and reserving GG individuals and eliminating AG individuals. After the selection and panning, the offspring can not have single-crown individuals.
Example 2 a new line with the antler crown trait was synthesized.
This example is the synthesis of a new line with the "deer horn" crown trait using pure breeder river chickens (line H) and other fast large single crown lines (line K).
Step 1 elimination of heterozygote individuals of H line
The method in example 1 was repeated to knock out AG individuals in the H-line population and to select GG-type individuals.
Step 2, crossing K-line cock and H-line hen
60 excellent K-line cocks meeting the requirements are selected to be hybridized with 600H-line hens which are selected by the aid of molecular markers to produce F1 generations.
Step 3F1 Individual Cross to give generation F2
Selecting cock and hen individuals meeting the requirement of F1 to perform crossing during the egg laying peak period to obtain F2 individuals.
Step 4, the heterozygote individuals of the F2 generation are eliminated
The method in example 1 was repeated to knock out AG individuals in the population of generation F2 and to select GG-type individuals.
Step 5, obtaining HK series by crossing
Utilizing F2 generation individuals subjected to molecular marker selection in the step 4 to carry out selfing to obtain the HK line (after the step 4 selection, non-antler crown individuals can not appear from the generation), and obtaining the HK new line with stable character heredity through continuous selection of three generations.
Sequence listing
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Claims (7)
1. The application of the molecular marker related to the chicken 'antler' crown in the genetic type panning of the chicken 'antler' crown is characterized in that the molecular marker is a gene sequence shown in SEQ.4 and/or SEQ.5, a mutation site is the 44 th nucleotide of the gene sequence, and a wild type allele sequence is shown in SEQ.4; the sequence of the mutant gene is shown in SEQ.No. 5.
2. The application of the molecular marker in the selection of the chicken 'deer horn' crown genotype, which is characterized in that the selection of the 'deer horn' crown genotype is carried out by extracting a sample blood DNA sample, carrying out multiplex PCR amplification on the sample blood DNA sample, carrying out an extension reaction after purifying a PCR product, sequencing the product, carrying out typing on a variation site, and then eliminating heterozygote individuals.
3. The application of the molecular marker in the genetic type panning of the antler crown of chicken as claimed in claim 1, wherein the genetic type panning method of the antler crown comprises the following steps:
step 1, eliminating heterozygote individuals of pure bred river-field chickens: removing AG individuals in the pure breed river-field chicken population by nucleotide detection of the variation sites, and selecting and reserving GG individuals;
step 2, hybridizing other fast large single-crown cocks and pure-breed Hetian chicken hens, namely hybridizing 60 excellent other fast large single-crown cocks meeting the requirements with 600 pure-breed Hetian chicken hens which are selected by the aid of molecular markers to produce F1 generations;
step 3, crossing the F1 individuals to obtain an F2 generation, namely the egg laying peak period, and selecting the cock and hen individuals meeting the requirement of F1 to cross to obtain an F2 generation of individuals;
step 4, eliminating heterozygote individuals of the F2 generation, namely eliminating AG type individuals in the F2 generation population through nucleotide detection of a mutation site, and selecting and reserving GG type individuals;
and 5, crossing to obtain a new line with the antler crown character, namely performing selfing on the F2 generation individuals subjected to molecular marker selection in the step 4 to obtain the new line with the antler crown character with stable character heredity.
4. The application of the molecular marker in the selection of the chicken 'antler' crown genotype according to claim 2 is characterized by comprising the following steps:
step 1: extracting sample DNA: extracting DNA from sample blood by phenol-chloroform extraction method;
step 2: typing of target sites:
(1) multiplex PCR amplification: mixing the forward primer and the reverse primer to obtain a mixed primer; adding sample blood DNA and nucleotide, adding water to constant volume to prepare a PCR system, and keeping the temperature at 95 ℃ for 3 min; 15s at 94 ℃ and 15s at 55 ℃ for 35 cycles; 30s at 72 ℃; carrying out amplification reaction under the amplification condition of 72 ℃ for 3min to obtain a PCR product;
(2) and (3) PCR product purification: the PCR product was purified using ExoI, FastAP and ExoI buffer;
(3) and (3) extension reaction: adding the purified PCR product into Snapshot Mix reagent and extension primer, and fixing the volume with water at 96 ℃ for 1 min; 10s at 96 ℃ and 5s at 52 ℃ for 30 cycles; carrying out extension reaction under the amplification condition of 30s at 60 ℃ to obtain an extension product;
(4) sequencing and typing: and (4) denaturing the extension product, sequencing the extension product by a sequencer, and eliminating heterozygote individuals according to the nucleotide of the target site.
5. The application of the molecular marker in the selection of the chicken 'antler' crown genotype according to claim 4 is characterized by comprising the following steps:
step 1: extracting sample DNA: extracting DNA from sample blood by phenol-chloroform extraction method;
step 2: typing of the site of interest
(1) Multiplex PCR amplification
Dissolving the forward primer and the reverse primer by using 1 × TE respectively until the concentration is 10pmol, uniformly mixing the forward primer and the reverse primer, and centrifuging to obtain a mixed primer; preparing a PCR system according to the following volume ratio: DNA 1-2. mu.L, 2. mu.L PCR mix 7.5. mu.L, mix primers 2. mu.L, add H2Supplementing O to 15 mu L, transferring the PCR system to a 96 micro-porous plate for PCR reaction, and carrying out 3min at 95 ℃; 15s at 94 ℃ and 15s at 55 ℃ for 35 cycles; 30s at 72 ℃; carrying out amplification reaction under the amplification condition of 72 ℃ for 3min to obtain a PCR product;
(2) PCR product purification
mu.L of the PCR product was taken, and ExoI 0.2. mu.L, FastAP 0.8. mu.L and ExoI buffer 0.7. mu.L were added thereto using H2Adding O to 7 mu L, mixing uniformly, and purifying the PCR product according to the purification conditions of 37 ℃ for 15min and 80 ℃ for 15min in sequence;
(3) extension reaction
Carrying out extension reaction on the purified PCR product, and preparing an extension reaction system according to the following components in volume ratio: after purification, 2. mu.L of PCR product, 1. mu.L of Snapshot Mix reagent, 1. mu.L of extension primer, and addition of H2Supplementing O to 6 mu L, transferring the obtained product to a 96 micro-porous plate for PCR reaction, and carrying out 1min at 96 ℃; 10s at 96 ℃ and 5s at 52 ℃ for 30 cycles; carrying out amplification reaction under the amplification condition of 30s at 60 ℃ to obtain an extension product;
(4) sequencing typing
Taking 1 mu L of extension product, adding 10 mu L of sample Hidi, denaturing at 95 ℃ for 3min, immediately performing ice water bath, and sequencing by a sequencer; individuals with the genotype of GG are selected and retained, and individuals with the genotype of the target site of AG are eliminated.
6. The application of the molecular marker in the panning of the chicken 'antler' crown genotype is characterized in that the sequence of the forward primer in the step (1) is shown as SEQ.No.1, and the sequence of the reverse primer is shown as SEQ.No. 2; the sequence of the extended primer in the step (3) is shown as SEQ.No. 3.
7. The application of the molecular marker in the panning of the chicken 'deer horn' crown genotype according to claim 6, wherein the method for extracting the DNA of the sample blood by the phenol-chloroform extraction method in the step 1 specifically comprises the following steps:
(1) putting 30 mu L of whole blood into a 1.5mL centrifuge tube, respectively adding 470 mu L of 1 xSET buffer solution, 12.5 mu L of 20% SDS and 6 mu L of 10mg/mL proteinase K, uniformly mixing, and putting in a 55 ℃ water bath for overnight;
(2) taking out a sample, putting the sample into a 1.5mL centrifuge tube, adding 500 mu L of saturated phenol, slightly shaking for 20min, and centrifuging at 10000rpm for 10 min;
(3) taking the supernatant, adding 500 μ L saturated phenol again, shaking gently for 20min, centrifuging at 10000rpm for 10 min;
(4) collecting supernatant, adding 500 μ L chloroform-isoamyl alcohol, wherein the volume ratio of chloroform to isoamyl alcohol is 23:1, shaking gently for 20min, centrifuging at 10000rpm for 10 min;
(5) taking the supernatant, adding 1mL of ice absolute ethyl alcohol with the temperature of-20 ℃, swinging back and forth to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out the ethyl alcohol;
(6) washing DNA once with 1mL of 75% ethanol, pouring off the ethanol, and drying in a drying oven at 50 ℃;
(7) after the DNA is completely dried, 300 mu L of sterilized double distilled water is added for dissolving, and the DNA is dissolved in a water bath kettle at 50 ℃ overnight;
(8) storing in a refrigerator at-20 deg.C for use.
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| CN107494410A (en) * | 2017-08-18 | 2017-12-22 | 四川农业大学 | A kind of method for the production depth shell color layer of green-shell egg that forms a complete set of |
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| CN108570506A (en) * | 2017-10-13 | 2018-09-25 | 江苏兴牧农业科技有限公司 | A kind of primer, kit and detection method for detecting rose comb locus gene |
| CN110079612A (en) * | 2019-05-14 | 2019-08-02 | 江苏省家禽科学研究所 | One kind SNP marker relevant to chicken weight, leg flesh weight and leg flesh muscle fiber trait and its detection method and application |
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| CN102041310B (en) * | 2010-09-13 | 2012-12-12 | 李宁 | Method for detecting rose cockscomb character |
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| CN107494410A (en) * | 2017-08-18 | 2017-12-22 | 四川农业大学 | A kind of method for the production depth shell color layer of green-shell egg that forms a complete set of |
| CN108570506A (en) * | 2017-10-13 | 2018-09-25 | 江苏兴牧农业科技有限公司 | A kind of primer, kit and detection method for detecting rose comb locus gene |
| CN108029634A (en) * | 2017-12-30 | 2018-05-15 | 江苏省家禽科学研究所 | A kind of high regularity is suitable for the producing method for seed of the cold yellow chicken breed system of fresh listing |
| CN110079612A (en) * | 2019-05-14 | 2019-08-02 | 江苏省家禽科学研究所 | One kind SNP marker relevant to chicken weight, leg flesh weight and leg flesh muscle fiber trait and its detection method and application |
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