CN108588204A

CN108588204A - Dove early sex PCR identification kits, application and method

Info

Publication number: CN108588204A
Application number: CN201810580648.9A
Authority: CN
Inventors: 尹兆正; 毛海光; 刘虹华; 温娅娅; 董信阳; 曹海月; 刘珂
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2018-09-28

Abstract

The present invention relates to a breeding pigeon early sex PCR identification kits and applications, and the method for carrying out early sex identification to dove using the kit.The kit includes mainly PCR amplification primer and PCR reaction reagents, and the PCR amplification primer sequence is as follows：Sense primer：5'‑TGCAGAAGCAATATTACAAGT‑3'；Downstream primer：5'‑AATTCATTATCATCTGGTGG‑3'；The beneficial effects are mainly as follows：The present invention provides the approach for carrying out early sex identification to dove by Molecular tools, compared with other methods such as turn over anus discriminating, laparoscopy, the detection of excrement steroids and caryogram detection, have many advantages, such as that accuracy is high, easy to operate, at low cost, small to animal injury, to pushing dove fine-variety breeding and industrialization to have a very important significance.

Description

Dove early sex PCR identification kits, application and method

(1) technical field

The present invention relates to a breeding pigeon early sex PCR identification kits and applications, and are carried out to dove using the kit The method of early sex identification.

(2) background technology

Dove, a kind of very common bird, all over the world raising extensively, Columbiformes, Columbidae.Dove is a kind of monomorphism bird, Its male and female appearance form difference is little, and unlike chicken, duck etc., other birds can distinguish from shape.Dove modes of reproduction is typical case " monogamy " system, if male and female ratio is improper, can cause dove group unstability, laying rate decline, be unfavorable for dove fine-variety breeding and Intensive management, be also required to go early in modern specialization egg dove production it is male stay mother, public dove is used for the production of conventional squab.Cause This Early Identification dove gender technology has most important theories and application value in dove fine-variety breeding and industrialization production.

(3) invention content

It is an object of the present invention to provide a breeding pigeon early sex PCR identification kits and applications, and utilize the kit pair The method that dove carries out early sex identification carries out early sex identification using Molecular tools to dove.

The technical solution adopted by the present invention is：

One breeding pigeon early sex PCR identification kits include mainly PCR amplification primer and PCR reaction reagents, the PCR Amplimer sequence is as follows：

Sense primer：5'-TGCAGAAGCAATATTACAAGT-3'

Downstream primer：5'-AATTCATTATCATCTGGTGG-3'.

Kit key of the present invention is the selected of primer sequence, and this field conventional reagent can be used in PCR reagent.

The present invention utilizes the special primer of CHD genes to the chromosome helix-destabilizing protein DNA combination genes of dove Homologous sequence on (chromo-helicase-DNA binding gene, CHD) carries out specific amplification and electrophoresis detection, from And the gender of dove is identified, obtain preferable result.

The CHD genes of birds are located on sex chromosome Z and W, the introne on two sex chromosome of the birds such as dove Length is different, causes length of the gene on two sex chromosome different, also demonstrate CHD genes for male and female judgement compared with It is accurate.Therefore, according to CHD gene introns, length is different on Z with W chromosomes, designs specific DNA molecular amplification and draws Object expands the different length segment of the gene, becomes the theoretical foundation of dove sex identification of the present invention.

The invention further relates to application of the kit in the identification of dove early sex.

The invention further relates to using the kit to dove carry out early sex identification method, the method includes：

(1) the dove feather root DNA that comes into being is extracted；

(2) DNA obtained using extraction carries out PCR amplification as template using PCR amplification primer；

The PCR amplification primer sequence is as follows：

Sense primer：5'-TGCAGAAGCAATATTACAAGT-3'

Downstream primer：5'-AATTCATTATCATCTGGTGG-3'

(3) pcr amplification product is detected into row agarose gel electrophoresis, if only amplification obtains the specific item of 1 447bp Band is then judged as male dove；If amplification obtains 2 specific bands of 447bp and 310bp, it is judged as female dove.

PCR response procedures are as follows：98 DEG C of pre-degeneration 2min；98 DEG C of denaturation 10s, 49.5 DEG C of annealing 30s, 72 DEG C of extension 30s, Totally 30 cycles；72 DEG C of extension 2min；4 DEG C of preservations.

The beneficial effects are mainly as follows：The present invention provides carry out early sex mirror to dove by Molecular tools Fixed approach, compared with other methods such as turn over anus discriminating, laparoscopy, the detection of excrement steroids and caryogram detection, it is accurate to have Property it is high, easy to operate, at low cost, small to animal injury the advantages that, it is particularly significant to pushing dove fine-variety breeding and industrialization to have Meaning.

(4) it illustrates

Fig. 1 is pcr amplification product electrophoretogram；

Fig. 2 is anatomy verification figure.

(5) specific implementation mode

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：

1 materials and methods

1.1 sample collection

Nascent dove of choosing carries the stipule 1~2 of hair follicle；It is put into 2ml centrifuge tubes, -20 DEG C of freezen protectives, for dividing Son experiment differentiates its gender.Make anatomy auxiliary identification simultaneously.

The processing of 1.2 feathers

1~2 feather with hair follicle for taking every plumage squeezes out the feather pulp at lower 1~2mm of root in EP pipes with tweezers, it After extract DNA.

1.3 DNA sample concentration mensurations

It is carried respectively with the detection of the agarose gel electrophoresis of ultramicron ultraviolet specrophotometer Nandrop2000 and 1.5% Take the concentration, purity and integrality of DNA.

1.4 design of primers and synthesis

2 pairs of primers below Selection utilization carry out specific amplification to dove gender-specific genes CHD respectively, and primer sequence is：

F：5'-TGCAGAAGCAATATTACAAGT-3',

R：5'-AATTCATTATCATCTGGTGG-3'.

1.6 PCR amplification

PCR amplification is carried out by template of extracted dove feather root DNA.PCR reaction systems 25uL：Template 5uL, above and below Swim primer each 2uL, PCR-Mix 12.5uL, sterile deionized water 3.5uL.PCR response procedures：98 DEG C of pre-degeneration 2min；98℃ It is denaturalized 10s, 49.5 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 cycles；72 DEG C of extension 2min；4 DEG C of preservations.Take 2.5uL PCR Amplified production is detected for 1.5% agarose gel electrophoresis.

The Morphological Identification of 1.7 sexual glands observation

10 doves of unknown gender are dissected with the Morphological Identification for making sexual gland observation after feather root DNA PCR detections, Sexual gland testis and female individuals sexual gland ovary according to male are criterion, then by PCR sex identifications result and solution It cuts open rear sexual gland observation result as coincidence rate to compare, determination rate of accuracy.

2 results

2.1 PCR product electrophoresis results

Using the combination primer, never corresponding segment, all females are amplified in the genomic DNA sample of the other dove of intellectual Individual shows as two bands, and all males show as a band, the result is shown in Figure 1.1,2,3,4, No. 5 are public dove (ZZ), are expanded Increase the specific band for 1 447bp or so；6,7,8,9, No. 10 are hen pigeon (ZW), amplify 2 spies of 447bp and 310bp Anisotropic band, amplification match with anatomy verification.Anatomical results are shown in Fig. 2.

According to the method described above, 200 pigeons, rate of accuracy reached 100% are identified.

Sequence table

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<120>Dove early sex PCR identification kits, application and method

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<213>Unknown (Unknown)

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aattcattat catctggtgg 20

Claims

1. a breeding pigeon early sex PCR identification kits include mainly that PCR amplification primer and PCR reaction reagents, feature exist In the PCR amplification primer sequence is as follows：

Sense primer：5'-TGCAGAAGCAATATTACAAGT-3'

Downstream primer：5'-AATTCATTATCATCTGGTGG-3'.

2. application of the kit described in claim 1 in the identification of dove early sex.

3. the method that early sex identification is carried out to dove using kit described in claim 1, the method includes：

(1) the dove feather root DNA that comes into being is extracted；

The PCR amplification primer sequence is as follows：

Sense primer：5'-TGCAGAAGCAATATTACAAGT-3'；

Downstream primer：5'-AATTCATTATCATCTGGTGG-3'；

(3) pcr amplification product is detected into row agarose gel electrophoresis, if only amplification obtains the specific band of 1 447bp, It is judged as male dove；If amplification obtains 2 specific bands of 447bp and 310bp, it is judged as female dove.

4. method as claimed in claim 3, it is characterised in that PCR response procedures are as follows：98 DEG C of pre-degeneration 2min；98 DEG C of denaturation 10s, 49.5 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 recycle；72 DEG C of extension 2min；4 DEG C of preservations.