CN101935710A

CN101935710A - Pigeon sex discriminating method

Info

Publication number: CN101935710A
Application number: CN201010507035.6A
Authority: CN
Inventors: 邢光东; 夏银; 茆骏; 李银
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2010-10-14
Filing date: 2010-10-14
Publication date: 2011-01-05
Anticipated expiration: 2030-10-14
Also published as: CN101935710B

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

A kind of dove sex discrimination method

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 by build, figure, turn over morphological method such as anus and carry out male and female and differentiate.Can wait by 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 male by ZZ() and ZW(female) form, and researched and developed in recent years thus into according to CHD(Chromo-helicase-DNA binding gene on the ZW karyomit(e)) the different PCR method of identifying the birds sex of expanding fragment length of gene.The PCR primer that is used for the birds sex identification at present mainly is Griffiths(1998) P2/P8 and the Fridolfsson(1999 of design) 2550F/2718R of design, 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 to extract from blood sample or many primaries feather pulps mostly.Above method weak point is, extracts animal blood sample or many primaries of allocation and easily birds or dove is 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 at 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 as follows:

A kind of dove sex discrimination method, by 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, by from the genomic dna template of dove feather pulp cell preparation, increase CHD gene fragment on Z and the W sex chromosome of round pcr, 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, 5 μ M upstream and downstream primers each 0.5 μ l, 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 as follows: (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) described feather pulp and the micro-zooblast genomic dna template of being equipped with prepared the centrifuge tube of liquid or 95 ℃ of insulations of PCR pipe 5 minutes, 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 at dove CHD gene conserved sequence specially, the amplification efficiency height, 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 genomic dna template preparation process is simple, and extraction cost is extremely cheap, makes the genomic dna template preparation of large-scale low-cost become possibility; Because genomic dna template preparation process is simple, 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 instrument 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 instrument, 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, 5 μ M upstream and downstream primers (SEQ ID NO.1, SEQ ID NO.2) each 0.5 μ l, 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.

Claims

1. dove sex discrimination method, it is characterized in that by 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, by from the genomic dna template of dove feather pulp cell preparation, increase CHD gene fragment on Z and the W sex chromosome of round pcr, 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, the described upstream and downstream of 5 μ M primer each 0.5 μ l, 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 as follows: (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) described feather pulp and the micro-zooblast genomic dna template of being equipped with prepared the centrifuge tube of liquid or 95 ℃ of insulations of PCR pipe 5 minutes, 16 ℃ of insulations 5 minutes; (4) in described centrifuge tube or PCR pipe, add Proteinase K; (5) 55 ℃ of insulations 2 hours or more than, 95 ℃ of insulations 10 minutes, 16 ℃ of insulations 5 minutes, centrifugal supernatant is directly as the dna profiling of PCR.

4. dove sex discrimination method according to claim 3 is characterized in that described micro-zooblast genomic dna template prepares liquid and 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 mixing solutions, and two kinds of reagent final concentrations are 0.1-2.0%.

5. dove sex discrimination method according to claim 3 is characterized in that described Proteinase K final concentration is 1.0-3.0mg/ml.