CN106577425A

CN106577425A - Panmixis parent tracing method for litopenaeus vannamei

Info

Publication number: CN106577425A
Application number: CN201611201011.1A
Authority: CN
Inventors: 黄文�; 陈廷; 任春华; 王艳红; 江晓; 胡超群
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2016-12-22
Filing date: 2016-12-22
Publication date: 2017-04-26
Anticipated expiration: 2036-12-22
Also published as: CN106577425B

Abstract

The invention discloses a panmixis parent tracing method for litopenaeus vannamei. The purposes of panmixis of litopenaeus vannamei and post parent tracing and identification are achieved in combination with a multiplication characteristic that spermatophore is still remained in the body of female litopenaeus vannamei after the litopenaeus vannamei spawns, and a litopenaeus vannamei microsatellite paternity test method which is mature currently. According to the method provided by the invention, the advantage that the female litopenaeus vannamei is high in successful mating efficiency during a production process is used, and the deficiency that the male does not mate with the female in directional mating and the position of the spermatophore is incorrect after mating is overcome; application of the method provided by the invention quickens the preparation of the full-sib families of the litopenaeus vannamei, and is beneficial to prompting the operation of litopenaeus vannamei molecular breeding.

Description

A kind of Litopenaeus vannamei random mating parent's retroactive method

Technical field

The invention belongs to genetic breeding field, and in particular to a kind of Litopenaeus vannamei random mating parent's retroactive method.

Background technology

Litopenaeus vannamei (Litopenaeus vannamei, also known as Penaeus vannamei) aquaculture is in China Fisheries economy Occupy larger proportion, be one of pillar industry of China's aquaculture.However, the genetic breeding work of China's Litopenaeus vannamei But fall behind very much, the close shrimp of high-quality relies on import, healthy, stable and sustainable development of this situation to China's prawn industry for a long time Exhibition brings larger hidden danger.Therefore, the basis and applied research for strengthening Litopenaeus vannamei genetic breeding field works, and will help In the unfavorable situation for improving China's Litopenaeus vannamei industry.

It is indispensable link in molecular breeding work to prepare the retrospective family full-sibs of a large amount of parents.In Fish In breeding work, can adopt in vitro artificial insemination method carry out, sperm and ovum can be obtained in vitro, Parent determine compared with Easily, and there is a highly developed technology of hastening parturition, therefore obtain the retrospective family full-sibs of substantial amounts of Parent and be easier to realize. However, Litopenaeus vannamei have embrace to knocking into the back, it is i.e. separate after mating, male shrimp spermatophore must be squeezed into female shrimp receptaculum seminis and could calculate real complete Conclusion of the business is matched somebody with somebody, and many tail hero shrimps and female shrimp priority mating often occurs, and only 1 tail can squeeze into the phenomenon of receptaculum seminis, artificial orientation Copulation occurs male shrimp not copulation again, or single tail copulation but spermatophore such as deflect at the unfavorable factor.In Litopenaeus vannamei reproductive process Unfavorable factor greatly limit the preparation process of family full-sibs, hinder prawn molecular breeding and Parents selection-breeding work Carry out.

The content of the invention：

The purpose of the present invention be for solve directional cross prepare family during deficiency, set up a kind of Litopenaeus vannamei with Machine copulation parent's retroactive method.

Litopenaeus vannamei random mating parent's retroactive method of the present invention, it is characterised in that comprise the following steps：

It is prepared by a, male shrimp DNA library：Litopenaeus vannamei Broodstock after sexual maturity idiophase is separately raised, and male shrimp is in male shrimp ponds Raise, female shrimp is raised in female shrimp ponds, and last is cut to swimmeret by all male shrimp in male shrimp ponds, then extract trip respectively The DNA of swimming foot, the male shrimp DNA library of composition；

B, close shrimp random mating：In morning 7-11, select the full female shrimp for extending to afterbody of gonad and be put in male shrimp ponds, Male shrimp and female shrimp pantogamy；

C, the spawning of female shrimp：In same day 18-21, female shrimp is pulled out, check whether receptaculum seminis squeezes into spermatophore, correctly beaten Enter the female shrimp of spermatophore, each female shrimp for correctly squeezing into spermatophore lays eggs in being put into single water body；

D, take spermatophore：Check whether female shrimp lays eggs in independent water body, immediately by female shrimp receptaculum seminis after finding to lay eggs Spermatophore is removed and cuts the swimmeret of the female shrimp that lays eggs；

E, parent review：The swimmeret DNA of the female shrimp of spawning is extracted, that is, obtains female parent gene group DNA；Remain in extracting spermatophore Thick white spermatium DNA, using the stable prawn micro-satellite primers of rich polymorphism, amplified production enter performing PCR amplification, And carry out matching identification with the result in male shrimp DNA library, the male shrimp DNA for matching is male parent gene group DNA, thereby determines that father This.

The invention has the beneficial effects as follows：The present invention remains on numerous in female shrimp body with reference to spermatophore after Litopenaeus vannamei spawning Feature is grown, at present more ripe prawn microsatellite paternity test method is added, Litopenaeus vannamei random mating, afterwards is realized Carry out the purpose that parent reviews identification.Present invention utilizes the female shrimp success mating efficiency high of the production process of Litopenaeus vannamei is excellent Point, overcomes the deficiency of orientation mating male and female not copulation or post-coitum spermatophore malposition, and the application of the inventive method will add The preparation of fast Litopenaeus vannamei family full-sibs, contributes to promoting the carrying out of prawn molecular breeding work.

Specific embodiment：

Following examples are that the present invention is further illustrated, rather than limitation of the present invention.

Embodiment 1：

1st, prepared by male shrimp DNA library：Litopenaeus vannamei after sexual maturity idiophase is separately raised, and male shrimp is raised in male shrimp ponds Support, female shrimp is raised in female shrimp ponds, by all 80 tail hero shrimps in male shrimp ponds, last is cut to swimmeret, in volume fraction 95% -20 DEG C of ethanol is preserved, and then extracts the DNA of swimmeret respectively, and individually (male 1,2,3 ... 80), the male shrimp DNA of composition for numbering Storehouse；

2nd, close shrimp random mating：In morning 7-11, select full 15 tail of female shrimp for extending to afterbody of gonad and be put into male shrimp Chi Zhong, male shrimp and female shrimp pantogamy；

3rd, female shrimp spawning：In same day 18-21,15 tails female shrimp is pulled out, check whether receptaculum seminis squeezes into spermatophore, obtain 10 Tail correctly squeezes into the female shrimp of spermatophore, and each female shrimp for correctly squeezing into spermatophore lays eggs in being put into single 1 barrel of water body；

4th, take spermatophore：After female shrimp drags for independent water body spawning, patrol once per 10-30 minutes, see whether female shrimp has produced Spermatophore in female shrimp receptaculum seminis is removed after finding spawning and cuts the swimmeret of the female shrimp that lays eggs, female shrimp swimmeret, essence by ovum immediately Pod is put in different test tubes, individually numbering (female 1,2,3 ... 10；Essence 1,2,3 ... 10), as far as possible collects thick white shape Spermatium, Liquid nitrogen storage；

5th, parent reviews：The swimmeret DNA of the female shrimp of spawning is extracted, that is, obtains female parent gene group DNA (female 1,2,3 ... 10)；The thick white spermatium remained in spermatophore is used to extract DNA, enters performing PCR using the prawn microsatellite sequence announced Amplification, and carry out matching identification with the result in the male shrimp DNA library of step 1, concrete grammar authorizes patent of invention " to enter with reference to China The multiple microsatellite identification system of row Litopenaeus vannamei Parentage determination and apply ZL201310340730.1 " described in method enter OK, the male shrimp DNA for matching is male parent gene group DNA, 8 tail of successful match hero shrimp genomic DNA (numbering：Male 6,8,9,20, 45,54,78,79).Obtain the retrospective family full-sibs of 8 groups of parents, parent identifies success rate, and (10 tails are effectively laid eggs up to 80% Female shrimp, 8 tail spermatophore DNA extraction successes).

Claims

1. a kind of Litopenaeus vannamei random mating parent's retroactive method, it is characterised in that comprise the following steps：

It is prepared by a, male shrimp DNA library：Litopenaeus vannamei Broodstock after sexual maturity idiophase is separately raised, and male shrimp is raised in male shrimp ponds Support, female shrimp is raised in female shrimp ponds, and last is cut to swimmeret by all male shrimp in male shrimp ponds, then extract swimming respectively The DNA of foot, the male shrimp DNA library of composition；

B, close shrimp random mating：In morning 7-11, select the full female shrimp for extending to afterbody of gonad and be put in male shrimp ponds, male shrimp With female shrimp pantogamy；

C, the spawning of female shrimp：In same day 18-21, female shrimp is pulled out, check whether receptaculum seminis squeezes into spermatophore, correctly squeezed into essence The female shrimp of pod, each female shrimp for correctly squeezing into spermatophore lay eggs in being put into single water body；

D, take spermatophore：Check whether female shrimp lays eggs in independent water body, immediately by the spermatophore in female shrimp receptaculum seminis after finding to lay eggs Remove and cut the swimmeret of female shrimp of laying eggs；

E, parent review：The swimmeret DNA of the female shrimp of spawning is extracted, that is, obtains female parent gene group DNA；What is remained in extracting spermatophore is white The DNA of the sticky spermatium of color, is entered performing PCR amplification using Litopenaeus vannamei micro-satellite primers, and is entered with the result in male shrimp DNA library Row matching identification, the male shrimp DNA for matching are male parent gene group DNA, thereby determine that male parent.