CN101935710B - Pigeon sex discriminating method - Google Patents
Pigeon sex discriminating method Download PDFInfo
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- CN101935710B CN101935710B CN2010105070356A CN201010507035A CN101935710B CN 101935710 B CN101935710 B CN 101935710B CN 2010105070356 A CN2010105070356 A CN 2010105070356A CN 201010507035 A CN201010507035 A CN 201010507035A CN 101935710 B CN101935710 B CN 101935710B
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Abstract
The invention belongs to the field of molecular biology and discloses a pigeon sex discriminating method comprising the following steps of: designing an upstream primer SEQIDNO.1 (Sequence Identification Number 1) and a downstream primer SEQIDNO.2 by comparing a 2550F/2718R primer amplified product of a pigeon CHD gene with a CHD gene sequence landed on NCBI (National Center for Biotechnology Information); amplifying CHD gene fragments (preparing a genomic DNA template from micro feather pulp cells) on Z and W sex chromosomes of a pigeon by PCR (Polymerase Chain Reaction); dyeing by EB (ethidium bromide); judging sex according to a band type through 1 percent of agarose gel electrophoresis; appearing two bands, namely that 628bp and 358bp are male; and appearing three bands, namely that 628bp, 420bp and 358bp are female. The invention combines a new preparation method of an animal cell genomic DNA template and a high-efficiency pigeon CHD gene fragment amplimer, and the once detection rate and the detection accuracy of the pigeon sex discrimination can reach 100 percent.
Description
Technical field
The invention belongs to biology field, relate to a kind of dove sex discrimination method.
Background technology
The early stage pairing of dove accurately is one of dove field practical technique.Dove is strict monogamy, if mispairing is not only had a fist fight between dove, and hen pigeon is not laid eggs.At present, except that indivedual kind doves can be according to feather from distinguishing the male and female that the dove of most kinds comprises squab, young dove, and even adult dove, can not be through build, figure, turn over morphological method such as anus and carry out male and female and differentiate.Can wait through build, figure and differentiate indivedual dove male and female of growing up though be rich in empirical keeper, accuracy rate is not high.
Birds sex chromosome is made up of ZZ (male) and ZW (female), and has researched and developed in recent years thus into the PCR methods of identifying the birds sex according to the expanding fragment length difference of CHD on the ZW karyomit(e) (Chromo-helicase-DNA binding gene) gene.The PCR primer that is used for the birds sex identification at present mainly is the 2550F/2718R that P2/P8 and the Fridolfsson (1999) of Griffiths (1998) design designs, and as the universal primer of all birds sex identification; The birds sex identification, the used genomic dna template of sex identification that comprises dove is from blood sample or many primaries feather pulps, to extract mostly.Above method weak point is, extracts animal blood sample or many primaries of allocation and is prone to birds or dove are damaged; Genome DNA extracting method adopts ordinary method mostly, and this method not only leaching process is loaded down with trivial details, and can not guarantee the disposable success of extracting genome DNA, and cost is higher relatively; PCR identifies and adopts the above two kinds of universal primer and other corresponding primer mostly with primer, but disposable recall rate is in fact not ideal.
Summary of the invention
The present invention seeks to provides a kind of dove sex discrimination method to the above-mentioned deficiency of prior art, and this method need not to get blood, only needs 1 veutro feather that contains a small amount of feather pulp can identify the dove sex, and accuracy rate is up to 100%.
Technical scheme of the present invention is following:
A kind of dove sex discrimination method; Through the 2550F/2718R primer extension product of comparison dove CHD gene and the CHD gene order (AY517718, AY517719) of NCBI login, design upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2, utilize this upstream and downstream primer; Through the round pcr CHD gene fragment on Z and the W sex chromosome that from the genomic dna template of dove feather pulp cell preparation, increases; Through EB dyeing, 1% agarose gel electrophoresis, gel imaging band somatotype is judged sex then; Two bands occur, promptly 628bp and 358bp's is male; Three bands occur, promptly 628bp, 420bp and 358bp's is female.
The PCR reaction system of described dove CHD gene fragment amplification is: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP0.4 μ l, each 0.5 μ l of 5 μ M upstream and downstream primers, Taq enzyme 0.05 μ l, genomic dna template 0.5 μ l; Reaction conditions is: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations.
Described dove genomic dna template extraction method is following: (1) allocation contains 1 on the nascent feather of dove veutro of a small amount of feather pulp, and gets the 0.2-5mg feather pulp in centrifuge tube or PCR pipe; (2) in centrifuge tube or PCR pipe, add micro-zooblast genomic dna template and prepare liquid; (3) with described centrifuge tube or 95 ℃ of insulations of PCR pipe 5 minutes that feather pulp and micro-zooblast genomic dna template prepare liquid are housed, 16 ℃ of insulations 5 minutes; (4) in described centrifuge tube or PCR pipe, add Proteinase K; (5) 55 ℃ are incubated 2 hours, and 95 ℃ are incubated 10 minutes, and 16 ℃ are incubated 5 minutes, and centrifugal supernatant directly is used as the dna profiling of PCR.
Described micro-zooblast genomic dna template prepares liquid and is got so that the volume ratio of 1:3-10 is mixed by A liquid and B liquid, and wherein said A liquid is Tris, (NH
4)
2SO
4, MgCl
2Mixing solutions, three kinds of reagent final concentrations are 0.1-2.0M; Described B liquid is Triton and β-coloured glaze base alcoholic acid mixed diluting liquid, and two kinds of reagent final concentrations are 0.1-2.0%.The solvent of A liquid, B liquid is distilled water.
The final concentration of described Proteinase K solution is 1.0-3.0mg/ml.
The volume sum that Proteinase K solution that adds during preparation genomic dna template and micro-zooblast genomic dna template prepare liquid is 10 μ l; It is 8.8 μ l that preferred described micro-zooblast genomic dna template prepares liquid long-pending, and the volume of described Proteinase K solution (10 mg/ml) is 1.2 μ l.
Beneficial effect of the present invention: dove CHD gene fragment amplification primer of the present invention is to design to dove CHD gene conserved sequence specially, and amplification efficiency is high, and the once success rate that the dove sex is differentiated can reach 100%; And this to primer when amplification CHD gene fragment is differentiated band as male and female, can amplify the non-CHD band of a 358bp, this band can be used as the reference band that the genomic dna template prepares the effect quality.The present invention has used a kind of new genomic dna method for preparing template, and this method prepares dna profiling from dove trace feather pulp cell, and not only sampling is simple, reduces the injury to dove; And the genomic dna template to prepare process simple, extraction cost is extremely cheap, makes the genomic dna template preparation of large-scale low-cost become possibility; Because it is simple that the genomic dna template prepares process, the genomic dna template of preparation is complete, and consume can not guarantee that the genomic dna template prepares disposable success.
Description of drawings
The PCR product electrophoresis result that the squab sex is differentiated among Fig. 1 embodiment 1.
Embodiment
Embodiment 1 squab sex is differentiated
1.1 squab feather pulp cell genomic dna template preparation
The newborn veutro feather of 7 days left and right sides of allocation squabs (containing a small amount of feather pulp) 1, and get 1mg left and right sides feather pulp in the PCR pipe;
Be equipped with and add micro-zooblast genomic dna template in the PCR pipe of feather pulp and prepare liquid 8.8 μ l;
Be equipped with PCR pipe that feather pulp and micro-zooblast genomic dna template prepare liquid put in the PCR appearance 95 ℃ 5 minutes, 16 ℃ 5 minutes, 55 ℃ 2 hours, 95 ℃ 10 minutes, 16 ℃ 5 minutes.And in first 16 ℃ of 5 minutes processes, suspend the PCR appearance, adding 1.2 μ l concentration is the Proteinase K solution of 10 mg/ml.Centrifugal supernatant directly is used as the dna profiling of PCR;
Wherein, above-mentioned micro-zooblast genomic dna template prepares liquid and is got so that the volume ratio of 1:7.8 is mixed by A liquid and B liquid, and A liquid is Tris, (NH
4)
2SO
4, MgCl
2Mixing solutions, three kinds of reagent final concentrations are 0.1M; Described B liquid is Triton and β-coloured glaze base alcoholic acid mixing solutions, and two kinds of reagent final concentrations are 0.1%, and the solvent of A liquid, B liquid is distilled water.
1.2 pcr amplification
Reaction system: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP 0.4 μ l, each 0.5 μ l of 5 μ M upstream and downstream primers (SEQ ID NO.1, SEQ ID NO.2), Taq enzyme 0.05 μ l, dna profiling 0.5 μ l; Reaction conditions: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations.
1.3 PCR product band somatotype (see figure 1)
EB dyeing, 1% sepharose 5-10V/cm electrophoresis, imaging system band somatotype.Female dove is three bands, and length is respectively 628bp, 420bp and 358bp; Male dove is two bands, and length is respectively 628bp and 358bp.
< 110>Jiangsu Province's research of agricultural science institute
< 120>a kind of dove sex discrimination method
<160>?2
<210>?1
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>CHD gene fragment amplification upstream sequence
<400>?1
aacgtggcaa?cagagtac 18
<210>?2
<211>?18
<212>?DNA
< 213>artificial sequence
<220>
< 223>CHD gene fragment amplification downstream sequence
<400>?2
gatccagtgc?ttgtttcc 18
Claims (4)
1. dove sex discrimination method; It is characterized in that through the 2550F/2718R primer extension product of comparison dove CHD gene and the CHD gene order of NCBI login; Design upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2 utilize this upstream and downstream primer, through the round pcr CHD gene fragment on Z and the W sex chromosome that from the genomic dna template of dove feather pulp cell preparation, increases; Through EB dyeing, 1% agarose gel electrophoresis; Judge sex according to banding pattern, two bands occur, promptly 628bp and 358bp's is male; Three bands occur, promptly 628bp, 420bp and 358bp's is female.
2. dove sex discrimination method according to claim 1 is characterized in that the PCR reaction system of described dove Z and the heterosomal CHD gene fragment amplification of W is: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP 0.4 μ l, each 0.5 μ l of the described upstream and downstream of 5 μ M primer, Taq enzyme 0.05 μ l, dove genomic dna template 0.5 μ l; The PCR reaction conditions is: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations.
3. dove sex discrimination method according to claim 1 and 2 is characterized in that described dove genomic dna method for preparing template is following: (1) allocation contains 1 on the nascent feather of dove veutro of a small amount of feather pulp, and gets 0.2～5mg feather pulp in centrifuge tube or PCR pipe; (2) in centrifuge tube or PCR pipe, add micro-zooblast genomic dna template and prepare liquid; (3) with described centrifuge tube or 95 ℃ of insulations of PCR pipe 5 minutes that feather pulp and micro-zooblast genomic dna template prepare liquid are housed, 16 ℃ of insulations 5 minutes; (4) in described centrifuge tube or PCR pipe, add Proteinase K; (5) 55 ℃ are incubated 2 hours, and 95 ℃ are incubated 10 minutes, and 16 ℃ are incubated 5 minutes, and centrifugal supernatant directly is used as the dna profiling of PCR; Described micro-zooblast genomic dna template prepare liquid by A liquid and B liquid with 1: the volume ratio of 3-10 is mixed, and wherein said A liquid is Tris, (NH
4)
2SO
4, MgCl
2Mixing solutions, three kinds of reagent final concentrations are 0.1-2.0M; Described B liquid is Triton and β-coloured glaze base alcoholic acid mixing solutions, and two kinds of reagent final concentrations are 0.1-2.0%.
4. dove sex discrimination method according to claim 3 is characterized in that described Proteinase K final concentration is 1.0-3.0mg/ml.
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| CN2010105070356A CN101935710B (en) | 2010-10-14 | 2010-10-14 | Pigeon sex discriminating method |
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|---|---|---|---|
| CN2010105070356A CN101935710B (en) | 2010-10-14 | 2010-10-14 | Pigeon sex discriminating method |
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| CN101935710A CN101935710A (en) | 2011-01-05 |
| CN101935710B true CN101935710B (en) | 2012-07-04 |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397091A (en) * | 2013-07-30 | 2013-11-20 | 华中农业大学 | Polymerase chain reaction (PCR) method for identifying sex of young pigeons |
| CN103468825B (en) * | 2013-10-14 | 2015-03-04 | 扬州大学 | Primer, kit and method used for goose sex identification |
| CN106070005A (en) * | 2016-06-08 | 2016-11-09 | 南京东晨鸽业有限公司 | A kind of high yield laying pigeon automatic sexing matching method |
| CN106434952A (en) * | 2016-11-02 | 2017-02-22 | 湖南农业大学 | Pigeon sex molecular identification method and primer pair used by method |
| CN108441550A (en) * | 2018-05-21 | 2018-08-24 | 湖南农业大学 | Primer combination, detection kit and its application of pigeon sex identification |
| CN110656182A (en) * | 2018-06-28 | 2020-01-07 | 深圳华大法医科技有限公司 | Composition for pigeon genotyping and application thereof |
| CN109913556A (en) * | 2019-01-08 | 2019-06-21 | 北京市农林科学院 | A kind of primer, kit and its method for Rapid identification dove gender |
| CN112094887A (en) * | 2019-06-17 | 2020-12-18 | 南京尧顺禹生物科技有限公司 | Gene identification optimization method applicable to large-scale sex identification of pigeons |
| CN114271237B (en) * | 2022-03-07 | 2022-05-27 | 南京农业大学 | Method for breeding long-short velvet self-sexing pigeons |
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| CN101333563B (en) * | 2008-07-23 | 2011-05-25 | 扬州大学 | Sex appraisal process for pigeon for meat |
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