CN111394473A - 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, the selection and purification of monogenic traits have three schemes, namely ① phenotype direct panning to gradually reduce the frequency of related recessive alleles in a population, ② test cross rejection of recessive alleles, ③ molecular marker assisted selection to directly reject corresponding alleles, wherein the scheme '①' requires a plurality of generations through the phenotype direct panning, and the recessive alleles always exist in the population and can not completely reject the recessive alleles, the scheme '②' theoretically can reject related recessive alleles through one generation, but errors are easy to occur due to the complexity of the process in the actual operation process, the scheme '③' has the characteristics of rapidness and simple process, and the premise is that the molecular markers which are completely linked with the traits or cause mutations related to the traits are known.
At present, a mode of removing wild allele from a river-field chicken/Binghuan chicken group or a group with the 'antler' crown character synthesized by taking the river-field chicken/Binghuan chicken as a material can only adopt a scheme '①' or a scheme '②'.
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 1.5m L centrifuge tube, respectively adding 470 mu L1 × SET buffer solution, 12.5 mu L20% SDS and 6 mu L10 mg/m L proteinase K, uniformly mixing, and putting in a water bath at 55 ℃ for overnight;
(2) taking out a sample, placing the sample in a centrifugal tube of 1.5m L, adding 500 mu L saturated phenol, slightly shaking for 20min, and centrifuging for 10min at 10000 rpm;
(3) taking the supernatant, adding 500 mu L saturated phenol again, shaking gently for 20min, and 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) collecting supernatant, adding 1m L ice anhydrous ethanol (-20 deg.C), rocking to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out ethanol;
(6) washing DNA once with 1m L75% ethanol, pouring off the ethanol, drying in a drying oven at 50 deg.C, (7) adding 300 μ L sterilized double distilled water to dissolve the DNA after it is completely dried, and standing overnight in a water bath at 50 deg.C to dissolve the DNA;
(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 with 1 × TE to 10pmol, mixing the forward primer and the reverse primer, centrifuging to obtain mixed primer, and preparing PCR system with DNA 1-2 μ L, 2 × PCR mix 7.5 μ L, mixed primer 2 μ L, and added H2O is supplemented to 15 mu L, the PCR system is transferred to a 96 micropore plate for PCR reaction according to the conditions of 95 ℃ for 3min, 94 ℃ for 15s, 55 ℃ for 15s, 35 cycles, 72 ℃ for 30s and 72 ℃ for 3minCarrying out amplification reaction under the amplification condition; 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
The 3. mu. L PCR product was taken and ExoI 0.2. mu. L0.8.8. 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
Carrying out extension reaction on the purified PCR product, and preparing an extension reaction system according to the following components in volume ratio of 2 mu L of the purified PCR product, 1 mu L of Snapshot Mix reagent, 1 mu L of extension primer and H2Supplementing O to 6 mu L, transferring the O to a 96 micro-porous plate for PCR reaction, and carrying out amplification reaction according to amplification conditions of 96 ℃ for 1min, 96 ℃ for 10s, 52 ℃ for 5s, 30 cycles and 60 ℃ for 30s 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 extension product, adding 10 mu L sample Hidi, denaturing at 95 ℃ for 3min, immediately performing ice water bath, sequencing by a sequencer, determining the genotype of a sample according to the sequencing result, selecting and reserving an individual with the genotype of GG, and eliminating an individual with the genotype of an objective site of AG.
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 1m L blood was drawn from the inferior vein of the chicken wings using a disposable syringe, injected into a 1.5m L autoclaved centrifuge tube containing about 200 μ L2% sterile EDTA anticoagulant, shaken gently, recorded wing number, and stored at-20 ℃ for future 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 1.5m L centrifuge tube, respectively adding 470 mu L1 × SET buffer solution, 12.5 mu L20% SDS and 6 mu L10 mg/m L proteinase K, uniformly mixing, and putting in a water bath at 55 ℃ for overnight;
(2) taking out a sample, placing the sample in a centrifugal tube of 1.5m L, adding 500 mu L saturated phenol, slightly shaking for 20min, and centrifuging for 10min at 10000 rpm;
(3) taking the supernatant, adding 500 mu L saturated phenol again, shaking gently for 20min, and 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) collecting supernatant, adding 1m L ice anhydrous ethanol (-20 deg.C), rocking to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out ethanol;
(6) washing DNA once with 1m L75% ethanol, pouring off the ethanol, drying in a drying oven at 50 deg.C, (7) adding 300 μ L sterilized double distilled water to dissolve the DNA after it is completely dried, and standing overnight in a water bath at 50 deg.C to dissolve the DNA;
(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 |
The prepared PCR manifold is subpackaged into 96-hole PCR plates and centrifuged, 2 mu L DNA samples are added into each hole, and the PCR plates are loaded into a PCR instrument after centrifugation
Specific amplification conditions were as follows:
(2) PCR product purification
After PCR amplification, 3 mu L PCR product was purified by ExoI and FastAP, mainly by removing the remaining primers in the reaction product with ExoI and removing the remaining DNTP in the reaction with FastAP
| 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
Taking 1 mu L extension product, adding 10 mu L sample Hidi, denaturing at 95 ℃ for 3min, immediately performing ice water bath, and loading a sequencer, wherein the parting result has two results, namely the sequencing result is respectively shown in figure 1 and figure 2, wherein figure 1 is the genotype sequencing result of a sample with the target site AG, and figure 2 is the genotype sequencing result of a sample with the target site 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 molecular marker related to the 'antler' crowns of the chickens is characterized by having a gene sequence shown in SEQ No.4 and/or SEQ No. 5.
2. A typing method of molecular markers related to chicken 'antler' crowns is characterized in that multiple PCR amplification is carried out after a sample blood DNA sample is extracted, extension reaction is carried out after PCR products are purified, sequencing is carried out on the products to type variation sites, then heterozygote individuals are eliminated, wherein the variation sites are 44 th nucleotides of amplified fragments, and wild type allele sequences are shown as SEQ.No. 4; the sequence of the mutant gene is shown in SEQ.No. 5.
3. The method for typing a molecular marker related to the crown of chicken "deer horn" according to claim 2, which comprises 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.
4. The method for typing a molecular marker related to the crown of chicken "deer horn" according to claim 3, which comprises 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 with 1 × TE to 10pmol, mixing the forward primer and the reverse primer, centrifuging to obtain mixed primer, and preparing PCR system with DNA 1-2 μ L, 2 × PCR mix 7.5 μ L, mixed primer 2 μ L, and added H2Supplementing O to 15 mu L, transferring the PCR system to a 96 micro-porous plate for PCR reaction, and carrying out amplification reaction according to the amplification conditions of 95 ℃ for 3min, 94 ℃ for 15s, 55 ℃ for 15s, 35 cycles, 72 ℃ for 30s and 72 ℃ for 3min to obtain a PCR product;
(2) PCR product purification
The 3. mu. L PCR product was taken and ExoI 0.2. mu. L0.8.8. 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 of 2 mu L of the purified PCR product, 1 mu L of Snapshot Mix reagent, 1 mu L of extension primer and H2O is supplemented to 6 mu L, and transferred to a 96 micro-porous plate for PCR reaction under the amplification conditions of 96 ℃ for 1min, 96 ℃ for 10s, 52 ℃ for 5s, 30 cycles and 60 ℃ for 30sCarrying out amplification reaction to obtain an extension product;
(4) sequencing typing
Taking 1 mu L extension product, adding 10 mu L sample Hidi, denaturing at 95 ℃ for 3min, immediately performing ice-water bath, sequencing by a sequencer, selecting and reserving individuals with the genotype of GG, and eliminating the individuals with the genotype of the target site of AG.
5. The method for labeling molecules related to the crown of chicken 'deer horn' as claimed in claim 4, wherein 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.
6. The method for typing chicken 'antler' crown-related molecular markers according to claim 4, wherein the method for extracting DNA from sample blood by phenol-chloroform extraction in the step 1 comprises the following steps:
(1) putting 30 mu L of whole blood into a 1.5m L centrifuge tube, respectively adding 470 mu L1 × SET buffer solution, 12.5 mu L20% SDS and 6 mu L10 mg/m L proteinase K, uniformly mixing, and putting in a water bath at 55 ℃ for overnight;
(2) taking out a sample, placing the sample in a centrifugal tube of 1.5m L, adding 500 mu L saturated phenol, slightly shaking for 20min, and centrifuging for 10min at 10000 rpm;
(3) taking the supernatant, adding 500 mu L saturated phenol again, shaking gently for 20min, and 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) collecting supernatant, adding 1m L ice anhydrous ethanol at-20 deg.C, swinging back and forth to precipitate DNA, centrifuging at 10000rpm for 10min, and pouring out ethanol;
(6) washing DNA once with 1m L75% ethanol, pouring off ethanol, and drying in a drying oven at 50 deg.C;
(7) after the DNA is completely dried, adding 300 mu L of sterilized double distilled water for dissolving, and putting the DNA in a water bath kettle at 50 ℃ overnight for dissolving the DNA;
(8) storing in a refrigerator at-20 deg.C for use.
7. The use of the molecular marker of claim 1 in the panning of chicken "deer horn" crown genotypes.
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