CN105219771B - The relevant molecular labeling of chicken frizzled feather character and its application - Google Patents
The relevant molecular labeling of chicken frizzled feather character and its application Download PDFInfo
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- CN105219771B CN105219771B CN201510756381.0A CN201510756381A CN105219771B CN 105219771 B CN105219771 B CN 105219771B CN 201510756381 A CN201510756381 A CN 201510756381A CN 105219771 B CN105219771 B CN 105219771B
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Abstract
The invention discloses the relevant molecular labeling of a chicken frizzled feather character and its applications, belong to biotechnology, the KRT gene linkage groups of chicken in E22C19W28_E50C23 regions（Genbank sequence numbers AADN03009152.1）There are one the bases of A56648 G56648 at 56648th bit base to replace, which causes SNP polymorphisms, replaces situation to make chicken marker assisted selection association analysis in the site base by using detection primer, judges chicken frizzled feather character.
Description
Technical field
The invention belongs to the molecular labeling preparing technical fields in poultry genetic engineering, and in particular to a kind of to be marked as chicken
It the molecular labeling relevant with chicken frizzled feather character of assisted Selection association analysis and its is applied in association analysis.
Background technology
In recent years, with the development of molecular genetics and biotechnology, the research in chicken molecular biology level is also more next
It is more.China is the country of an exquisite cuisines, the favor to chicken, derived from the eating habit formed over the past thousands of years, poultry product
Focus on product External Characters based on fresh and alive in consumption market, it is desirable that product has buys phase, such as feather color, shape well.
Though these characters, due to the consumption habit that China different regions are formed for a long time, are had led to certain without economical value
The preference of character, the price purchase for preferring out higher, so as to be formed indirect economic characters.
Chicken mainly has three flat hair, Si Mao and frizzled feather feather types, and frizzled feather chicken is similar to the curly hair of people, and not only appearance is beautiful,
Also there is certain heat resistance.At present, the selection and breeding of frizzled feather chicken breed and merchandized handling are faced with some technical barriers, main problem
It is the holding of frizzled feather character.Since frizzled feather character is controlled by codominant gene, homozygous frizzled feather genotype and heterozygosis partly turn over type and are difficult
Pass through phenotype selection and breeding so that frizzled feather chicken group is difficult to purify.So using conventional breeding means, the period of selection is long, into postponing
Slowly.In addition, to purifying frizzled feather group, need to detect heterozygous individual by the method for test cross.It is carried out when to a certain test individual
During test cross, when need to observe that the offspring of 5 or more is frizzled feather, the assurance for just having 95% assert that test individual genotype is aobvious
Property it is homozygous.It needs to carry out a large amount of test cross in this way, not only extends the period of selection and breeding, also cause very high financial resources, manpower burden.Cause
This, separation clone influences the new gene of the frizzled feather character of chicken and finds important molecular labeling for chicken marker assisted selection, right
Accelerate the selection and breeding of chicken new varieties, the development for accelerating China's poultry husbandry is extremely important.Molecular labeling (DNA
Molecular marker) technology development it is perfect, for poultry improvement provide new means, utilize molecular marker assisted selection
(Marker Assisted Selection, MAS) carries out assisted Selection to genotype, can make full use of phenotype, pedigree and something lost
The information of label is passed, compared with only utilizing the conventional seed choosing method of phenotype and pedigree information, there is the information content of bigger, superiority
Clearly.Therefore, marker assisted selection is increasingly taken seriously, and is constantly applied in the practice of poultry breeding.
At present, it has found in the selection and breeding of chicken and applies some DNA molecular markers, such as Chicken and fish fishy smell（FMO3）Gene, green shell
（SLCO1B3) gene and short and small (dw) gene etc..
With other several genetic markers --- compared with morphology label, biochemical biomarker, cytological marker, DNA molecular
Label has lot of advantages：Abundant, label the quantity that makes a variation in the genome of chicken is almost unlimited；Chicken Objective is not influenced
The expression of shape, does not have linkage relationship, being capable of easy detection label and the correlation of chicken correlated traits soon with bad character.Extensively
It is general to be applied to the side such as genetic breeding, genomic mapping, the assignment of genes gene mapping, the discriminating of species affiliation, gene pool structure, gene cloning
Face.
Invention content
KRT75 related genes play a significant role in hair and nail development, but its functional study is not also very thoroughly
Thorough, the present invention is had found by the association analysis between character between gene and character by finding the variant sites in the gene
Relationship is an important means for studying gene function and the basis that assisted Selection is marked, and based on this, the present invention provides
As chicken marker assisted selection and the relevant molecular labeling of chicken frizzled feather character, and the molecular labeling is auxiliary as the label of chicken
Help the application of selection.
The invention is realized by the following technical scheme：
The relevant molecular labeling of chicken frizzled feather character, its nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
More specifically, in SEQ ID NO：Base at 117th bit base of sequence shown in 1 there are one A117-G117 is replaced
It changes, which causes SNP polymorphisms.
It is wherein used to detect SEQ ID NO：Base at 117th bit base of sequence shown in 1 there are one A117-G117 is replaced
The DNA sequence dna for the primer pair changed is as follows：
Forward primer：5 '-TGATGACCTACGGAACAACC-3 ',
Reverse primer：5′-ATGGCAATGGATGGCTGA -3′.
The aforementioned relevant molecular labeling of chicken frizzled feather character is built as follows：
A extracts the genomic DNA of chicken to be measured；
Design primer in b chicken E22C19W28_E50C23KRT gene linkage groups region.
C carries out PCR amplifications using genomic DNA as template, with design primer pair, obtains PCR amplified productions, PCR productions
Object direct Sequencing, is compared sequencing result, correlation analysis verification is carried out to mutational site and frizzled feather character, on finding
State primer pair；
D is expanded using above-mentioned primer, measures product nucleotide sequence.
The relevant molecular labeling of chicken frizzled feather character of the present invention can be used for being applied to mark with chicken frizzled feather correlated traits assisting
In the association analysis of selection.
The present invention also provides a kind of frizzled feather genetic homozygous (AA) assisted in identification frizzled feather chicken, frizzled feather genetic heterozygosis
The method of sub (AG) peaceful hair gene homozygote (GG), includes the following steps：
A extracts the genomic DNA of chicken to be measured；
B carries out PCR amplifications using genomic DNA as template, with the primer pair, obtains PCR amplified productions；
Identification PCR amplification direct Sequencings are sequenced according to PCR products in c, product nucleotide sequence are measured, so as to judge to be
Chicken to be measured is the homozygote that candidate is frizzled feather genetic homozygous or the peaceful hair gene of frizzled feather genetic heterozygosis.
The present invention protects frizzled feather genetic homozygous (AA), frizzled feather gene of the primer pair in auxiliary identifies frizzled feather chicken miscellaneous
Application in zygote (AG) peace hair gene homozygote (GG).
The primer pair can be used for preparing frizzled feather genetic homozygous, frizzled feather genetic heterozygosis in auxiliary identification frizzled feather chicken
The peaceful homozygous kit of hair gene.
The primer pair, the method or the kit are used equally for frizzled feather chicken breeding.If you need to frizzled feather chicken into
Row selection and breeding are to improve purity, which can be used to the variation to frizzled feather site into line trace, so as in selection and breeding offspring
Middle holding frizzled feather character.
The present invention also protects a kind of frizzled feather chicken breeding method, is carried out using the frizzled feather genetic homozygous in frizzled feather chicken
Breeding.
The present invention can make up the deficiency of conventional breeding methods, shorten breeding cycle, improve the accuracy of breeding.The present invention
The advantages of：(1) it establishes on the basis of frizzled feather gene finely positioning, 1 SNP site, the label is found in localization region
It is significantly associated with frizzled feather character, infers that individual is more than 94% for the accuracy of frizzled feather according to marker genetype；(2) not by gender
Limitation；(3) present invention is it can be inferred that individual phenotype and genotype, can early select early to eliminate, can contract in individual chick stage
Short breeding time accelerates breeding process, saves feeding cost.
Description of the drawings
Fig. 1：It is chicken E22C19W28_E50C23 regions KRT gene linkage groups DNA fragmentation used for positioning in the present invention.Institute
Primer sequence is marked (base in bracket for mutation) with box.
Fig. 2 is chicken E22C19W28_E50C23 regions KRT gene linkage groups DNA fragmentation sequencing used for positioning in the present invention
Figure, frequency of genotypes AA.
Fig. 3 is chicken E22C19W28_E50C23 regions KRT gene linkage groups DNA fragmentation sequencing used for positioning in the present invention
Figure, genotype AG.
Fig. 4 is chicken E22C19W28_E50C23 regions KRT gene linkage groups DNA fragmentation used for positioning in the present invention
Sequencer map, genotype GG.
Specific embodiment
The invention will be further described in the following with reference to the drawings and specific embodiments, these embodiments are only used for illustrating
The bright present invention, without forming any restrictions to the scope of the present invention.
Embodiment 1, chicken E22C19W28_E50C23 regions chicken KRT gene linkage group A117G mutational sites specificity
Design of primers
It is the famous frizzled feather chicken breed in China to repair water Huang plumage Gallus domesticlus brisson, because frizzled feather gene is dominant gene, so in group still
The heterozygote of recessive gene is carried in the presence of part.The offspring generated between frizzled feather genetic homozygous, overmolded character do not detach,
All it is frizzled feather.About 1 can be generated between frizzled feather genetic homozygous and frizzled feather genetic heterozygosis:1 frizzled feather genetic homozygous and frizzled feather
Genetic heterozygosis, so as to which non-frizzled feather gene passage be made down.The offspring generated between frizzled feather genetic heterozygosis, it may appear that flat hair
Individual.
First, discovery polymorphic SNP
Find that one marks with the significantly associated SNP of frizzled feather character in sequencing.In E22C19W28_E50C23 regions chicken
KRT gene linkage groups（Genbank sequence numbers AADN03009152.1）There are one A56648- at 56648th bit base
The base of G56648 is replaced, which causes SNP polymorphisms.
2nd, the design of primer pair
Primer is designed in chicken E22C19W28_E50C23 KRT gene linkage groups region, using genomic DNA as template, is used
Above-mentioned primer pair carries out PCR amplifications, obtains PCR amplified productions, and sequencing result is compared in PCR product direct Sequencings,
Analysis is associated to mutational site and frizzled feather character and is verified, position is mutated so as to obtain a pair for detecting chicken frizzled feather A117G
The PCR primers of point.
Sense primer F：5′-TGATGACCTACGGAACAACC-3′（SEQ IDNO.1）
Downstream primer R：5′-ATGGCAATGGATGGCTGA -3′（SEQ ID NO.2）.
Sense primer is by 20 base compositions, the KRT gene linkage groups of corresponding E22C19W28_E50C23 regions chicken
（Genbank sequence numbers AADN03009152.1）56745-56764；Downstream primer is corresponding by 18 base compositions
The 56349-56366 bit base sequences of AADN03009152.1.Primer amplification segment length 416bp.
Embodiment 2, the amplification of frizzled feather gene PCR and sequencing for repairing water Huang plumage Gallus domesticlus brisson
260 are repaiied water Huang plumage Gallus domesticlus brisson：Purchased from Xiushui county Jiangxi province.
1st, genomic DNA is extracted
Each sample wing venous blood sampling, extracts high salt method genomic DNA.
Detailed process is as follows：
(1) solution is configured, for the extracting genome DNA of chicken to be measured
Solution A：50mM TrisCl (pH 8.0), 5mM EDTA (pH 8.0), 20% SDS, room temperature preservation.It is molten
Liquid B：Chloroform:Isoamyl alcohol (24:1), shading preserves.
1) 100 μ l chicken bloods are taken to 1.5 mL centrifuge tubes
2) add 600 μ L solution As to 1.5 mL centrifuge tubes.
3）Add the ProK (20 mg/mL) of 10 μ L, abundant mixing into each 1.5 mL centrifuge tubes.
4）55 DEG C of water-bath digestion are overnight.
5）Isometric Tris saturated phenols are added in into each centrifuge tube, shake mixing 15min, 12000r/min centrifugation
10min。
6）Supernatant liquor is taken out, is fitted into new centrifuge tube, repeats the above steps.
7）Supernatant liquor is taken, adds in isometric phenol:Solution B (1:1) mixing 10min, 12000r/min are centrifuged
10min。
8）Supernatant liquor is taken to add in isometric solution B, mixing 10min centrifugations 12000 r/min, 10min.
9）Take supernatant liquor add in 2 times of volumes ice ethyl alcohol (about 1 mL) again plus 1/10 volume about 60 μ L NaAc water
Yawing moves, it is seen that cotton-shaped DNA sample occurs.
10）Sample is placed in -20 DEG C of 30 min of freezing, takes out or chooses DNA or centrifugation, 12000r/min, 10min, DNA
It is sunken to tube bottom.
11）DNA is washed with 75% ethyl alcohol, after shaking washing, centrifuges 12000 r/min, 5-10min.
12）75% ethyl alcohol is discarded, after natural drying, adds in appropriate (about 100 μ L) TE dissolvings.
13) (genomic DNA) is preserved for -20 DEG C.
2nd, using genomic DNA as template, the primer pair designed with embodiment 1 carries out PCR amplifications, obtains PCR amplifications
Product.PCR reaction systems (25 μ l)：2.5 μ l 10 × Taq DNA polymerase buffers (500mmol/L KCl,
100mmol/L TrisCl, 15mmol/L MgCl2), (concentration of ATP, TTP, GTP, CTP is 1 μ l dNTPs
2.5mmol/L), 1 μ l primers（0.5μM）, 0.25 μ l 2.5U/ μ L Taq DNA polymerases, 60ng templates.PCR reaction conditions：
95 DEG C of 5min, 94 DEG C of 30 s, 56 DEG C of 30s, 72 DEG C of 30s, 33 cycles, 72 DEG C of 10min, 4 DEG C of preservations.
3rd, PCR amplified productions are sequenced
PCR product is sequenced using double deoxidation chain termination method on automatic dna sequencer, and sequencing is by Nanjing gold
It completes Si Rui bio tech ltd.Sequencing result shows：The nucleotide sequence of the PCR amplified productions of AA genotype individuals
Shown in Fig. 2；Shown in nucleotide sequence Fig. 3 of the PCR amplified productions of AG genotype individuals；The PCR of GG genotype individuals expands
Increase production object nucleotide sequence Fig. 4.
SEQ ID NO：1
〈210〉：1
〈211〉:20
〈212〉：DNA
〈213〉：Artificial sequence
〈400〉: 1
TGATGACCTA CGGAACAACC 20
SEQ ID NO：2
〈210〉：1
〈211〉:18
〈212〉：DNA
〈213〉：Artificial sequence
〈400〉: 1
ATGGCAATGG ATGGCTGA 18
SEQ ID NO：3
〈210〉：1
〈211〉:416
〈212〉：DNA
〈213〉：Frizzled feather chicken
〈400〉: 1
TGATGACCTA CGGAACAACC AGAGTGAGAT AGCTGAGCTG AACCGGATGA TCCAGAAGCT 60
GCAGTGTGAA TCAGATAACG TGAAGAAACA GGTAGGTGAA GAGCTGAGTG CTGCACAGGA 120
GGCAGTGTGC AAAGGAAGCC CCAGCTCCTG CCTGGATGTG TCATCACGCT TCTCTAGGTG 180
GCAGGTTTAT TTCCACCTAG GAGAATCCAG TTGGTTTGGC TCTACCAGAA ATAATGGTGG 240
CTGCTTGTTT GCTACAGAGC AGCCAACACA TCCTTTCCCT TCATCCCCGA TTGCATGGCT 300
ACTGCAGGTG CCCAATCAAA ATCTAGGTTT CCTCAGCAGT TTCTAGCTGA GAAACAAGGG 360
GGGAAACTTG CAGCCCTGCC AAGCTCTGTG TTGATCTCTC AGCCATCCAT TGCCAT 416
Claims (3)
1. the relevant molecular labeling of chicken frizzled feather character, it is characterised in that：Nucleotide sequence as shown in sequence 1 and 2,
There are one the bases of A117-G117 at 117th bit base of sequence shown in sequence 3 to replace, wherein for 3 institute of detection sequence
Show that the DNA sequences for the primer pair replaced at the 117th bit base of sequence there are one the base of A117-G117 are as follows：
Forward primer：5 '-TGATGACCTACGGAACAACC-3 ',
Reverse primer：5′ -ATGGCAATGGATGGCTGA -3′.
2. the application of the relevant molecular labeling of chicken frizzled feather character described in claim 1, it is characterised in that：It is turned over applied to chicken
In the association analysis of hair correlated traits marker assisted selection.
3. a kind of auxiliary identifies that the frizzled feather gene that frizzled feather genetic homozygous, genotype that the genotype in frizzled feather chicken is AA are AG is miscellaneous
Zygote or the homozygous method of flat hair gene that genotype is GG：
A extracts the genomic DNA of chicken to be measured；
B designs primer to chicken E22C19W28_E50C23KRT gene linkage groups region；
C carries out PCR amplifications using genomic DNA as template, with design primer pair, obtains PCR amplified productions, PCR products are straight
Sequencing is connect, sequencing result is compared, correlation analysis verification is carried out to mutational site and frizzled feather character, it will so as to obtain right
Seek primer pair described in 1；
D. with the primer pair described in claim 1, using the genomic DNA of chicken to be measured as template, PCR amplifications is carried out, measure production
Object nucleotide sequence, so as to judge that chicken to be measured is that frizzled feather genetic homozygous or frizzled feather genetic heterozygosis or flat hair gene are homozygous
Son.
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