CN102630616B - Method for improving ratio of female fish in cynoglossus semilaevis cultured fingerlings - Google Patents
Method for improving ratio of female fish in cynoglossus semilaevis cultured fingerlings Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention relates to a method for improving the ratio of female fish in cynoglossus semilaevis cultured fingerlings. The method comprises the following steps of: 1) screening male parent fish; 2) performing artificial propagation and family establishment; 3) cultivating fish fries with different families and detecting the ratio of male and female fish; and 4) cultivating a high-female and fast-growing family and propagating, wherein a genetic marker used in the step 1) is SChen-SSR2. According to the invention, a method for identifying cynoglossus semilaevis genetic female fish and genetic male fish is established by the SChen-SSR2 marker, and then a method for improving the ratio of female fish in cynoglossus semilaevis cultured fingerlings is established by breeding generations fromthe high-quality normal male fish or establishing a cynoglossus family with a high female ratio bred by the families. The method provided by the invention can be used for solving the most common problems of too high ratio of male fish and too low ratio of physiological female fish in the cultured populations, and thus the method is of great importance in the scale production and cultivation of high-quality cynoglossus semilaevis fingerlings, the improvement of cultivation yield and economic benefit, and the promotion of continuous and rapid development of the cynoglossus semilaevis cultivation industry.
Description
Technical field
The invention belongs to the Sex Control field in the marine fish culture, be specifically related to a kind of method that improves cynoglossus semilaevis cultivation seed raun ratio.
Background technology
Cynoglossus semilaevis (Cynoglossus semiliaevis) is the distinctive a kind of famous and precious economic seawater fish of China, belongs to coastal waters warm water demersal fishes, and China coast all has distribution, take the Huanghai Sea, the Bohai Sea as many.Cynoglossus semilaevis is welcome by consumers in general deeply owing to its delicious flavour, fine and tender taste, nutritious, its market value is high, and the cultivation prospect is boundless.As one of principal item of China's marine fish culture, Cynoglossus semilaevis year cultured output reach at present more than 10,000 ton, calculate by average 120 yuan/500 grams of the market price, the cynoglossus semilaevis cultivation industry at present annual output value reaches more than 2,400,000,000 yuan.
In view of the cultivation potentiality of Cynoglossus semilaevis and potential economic and social benefits, in recent years, China scientific worker has carried out large quantity research to Cynoglossus semilaevis genital regulating and natural spawning technology, 2003-2007 mainly adopts the method for Artificial Control illumination and water temperature to stimulate the Cynoglossus semilaevis parent population ripe, and induce its natural spawning in indoor cement pit, obtain Cynoglossus semilaevis fertilized egg, and produced semi-smooth tongue sole offspring breed (Liu Xuezhou etc., 2006).This Cynoglossus semilaevis seedling raising manners, basically be with artificially controlling temperature, control light stimulus parent population maturation and carry out natural spawning and natural insemination in culturing pool that it is not too high to exist controllability, the Gunther parent fish spawning rate is lower, the low deficiency that waits of fertilization rate is unfavorable for planned arranging production.Particularly this natural spawning mode can not obtain the unfertilized egg of Cynoglossus semilaevis, is not suitable for cynoglossus semilaevis gynogenesis and induces and family foundation, has affected the process of semi-smooth tongue sole offspring breed propagation in scale and prevalent variety cultivation.Therefore; experts and scholars have carried out the research of tongue sole induced spawning technology subsequently; set up the technology that makes parent population grow and ovulate synchronously by the injection hormone; can obtain in a large number unfertilized egg by the artificial induced spawning technology; thereby be that important foundation (Yang Jingfeng has been established in the foundation of Cynoglossus semilaevis artificial gynogenesis and family; Chen Songlin etc., 2010), for Cynoglossus semilaevis scale breeding, sex control and prevalent variety cultivation provide technological means.
Cynoglossus semilaevis cultivation was put into practice and be studies show that there were huge difference in Cynoglossus semilaevis female individuals and male in growth rate in recent years, and its female individuals than the large 3-4 of male doubly.After the Cynoglossus semilaevis fry cultivated through 1 year half, female individuals can be grown the 500-750 gram, and male then only has the 50-150 gram.Because it is slow that male was grown, affected the quality of marketable fish, reduced the cultured output of Cynoglossus semilaevis, increase aquaculture cost, thereby had a strong impact on the popularization of semi-smooth tongue sole offspring breed and the development of aquaculture industry.Particularly nearest, (2010) such as aspect mountains studies show that to propagate artificially at Cynoglossus semilaevis and exist male pseudo-milter on female in a small amount of heredity, the physiology in the colony, and the growth rate of these pseudo-milters is starkly lower than raun.Simultaneously, investigation shows, in some Cynoglossus semilaevis nursery and plant, Cynoglossus semilaevis propagates that the ratio of milter reaches 70-90% in the colony artificially, these milters are failed to grow up, and the ratio of the raun that can grow up only is 10-30% (Chen Songlin etc.), has had a strong impact on raiser's enthusiasm, has affected the development of industry.Therefore, the method for milter ratio, raising raun ratio is extremely important for the cultured output and the product quality that improve Cynoglossus semilaevis in the searching minimizing cultured population.If the physiology raun ratio in the cynoglossus semilaevis cultivation colony can be brought up to 50% or more than, namely on present cultivation level with cynoglossus semilaevis cultivation colony in the ratio of raun improve about 20%, after carrying out spread, will increase by the economic benefit of hundred million yuan of 2-4 every year.
Find out the high reason of physiology milter ratio in the common seed that Cynoglossus semilaevis propagates artificially, must set up Cynoglossus semilaevis genetic sex and physiology sex authenticate technology, find out the source of milter in the common seed of Cynoglossus semilaevis, and then take measures, reduce the quantity of male fry, improve the ratio of female fry.Therefore at first must screen the special molecular labeling of sex.The sex specific molecular marker is the very potential technological means of identifying sex.But the at present research of relevant fish sex linkage molecule mark and rapid identification method was carried out a few fish, screened the special dna fragmentation of chinook Y chromosome such as the Devlin (1994) in Canadian West Vancouver laboratory; Griffiths etc. (2000) have found two male special AFLP marks in three vertebra sticklebacks (Gasterosteus aculeatus L.).But, when being applied to other two kinds of sticklebacks, but can not correctly identify its sex.Rachael etc. (2003) have compared the Y linkage collection of illustrative plates of 4 kinds of salmons (arctic salmon, Atlantic salmon, brown trout and rainbow trout), have found respectively the AFLP mark of 2 and 6 gunther sex-linkeds in arctic salmon and rainbow trout.But these marks have the specificity of planting, so range of application is narrower.Because the fish sex differentiation is comparatively original, male and female sex chromosome difference is little, thereby the evaluation of its genetic sex is comparatively difficult.Aspect the screening of Cynoglossus semilaevis sex specific molecular marker, Chen etc. (2007) are separated to the female specific AFLP mark of Cynoglossus semilaevis, after converting it into the SCAR mark, set up the PCR method of identifying Cynoglossus semilaevis genetic sex, for the Cynoglossus semilaevis genetic sex evaluation provides new technological means, but the special microsatellite marker of relevant employing sex is female in the cynoglossus semilaevis cultivation colony by detecting, the ratio of milter and pseudo-milter, and the method that how to improve raun ratio in the cynoglossus semilaevis cultivation colony, there is not yet so far report both at home and abroad.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves raun ratio in the cynoglossus semilaevis cultivation fry, thereby solve the difficult problem that the raun ratio is excessively low, cultured output is not high in the present cynoglossus semilaevis cultivation colony.
The present invention at first adopts sex chromosome linkage molecule mark that breeding is screened with parent population, therefrom select high-quality normal male parent population, and then adopt the male parent population of high-quality to set up family, by the physiology sex ratio of more different family fries and the growth rate of different family fries, the family that therefrom filters out physiology raun ratio height, fast growth cultivates and breeds as superior families, thereby overcome the low problem of raun ratio in the cultured population, and then improve physiology raun ratio, raising growth rate and cultured output in the cynoglossus semilaevis cultivation seed.
Technical scheme of the present invention is as follows:
1) screening of the male parent population of Cynoglossus semilaevis: the male parent population of Cynoglossus semilaevis is carried out genetic sex identifies, with genetic sex be male, simultaneously the physiology sex also be male parent fish selection out;
Describedly the male parent population of Cynoglossus semilaevis is carried out genetic sex identifying, is to increase by the primer that designs from the Cynoglossus semilaevis sex-specific marker to identify;
A kind of sex specific molecular marker is the chain microsatellite marker SChen-SSR2 of Cynoglossus semilaevis sex chromosome (male and female consensus sequence and female characteristic sequences):
Male and female have fragment sequence (274bp): SEQ ID NO:1
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGGCCTATTTGTAATATTTCAGTACTAGATGTATCCCTACAAATTGTGTTTAGAGGTAAGCATTTAAATAGTGGTTTTTATTGACAAATATGCAGATTGGATGTCGGGAGGTCGTCGTGCAGCTCATATAACTAGACATTATCGCCTGTTGAGTTTGTGGCTAGAAGCCAACTGGAAGGTTAGATTGTAGAAAATTGTTTTTTTTTTTTTTTGTCGTG
TGAGTACAACAC TAGTCCAACTAGTGC；
Female peculiar fragment sequence (302bp): SEQ ID NO:2
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGACAATTTTGTAATATTTCAATCCTAGATGTATCCCTATAAATTGTGTTTAGAGGTAAGCATTTAGATGTGGTTTAGAAGTGGTTTTTATTGTATTGTATTGTATTGTATTGTATTGTATGCAGCTTAGCTGTCAGGAGGTCGTCGTGTGGTTCATTTAACTAGACATTATCACCTGCTGAGTTTGTGGCCAGAAGCCAACTGGAAGGTTAGATTGTAGAAAACAGTTTATTTTTATGTGTTGTG
TGAGTACAACACTAGTCCAACTAGTGC；
The primer that designs from SChen-SSR2 is Primer 1:(SEQ ID NO:3:CAG TGTGTGTTTATTTAAAGGTTAGTTGGGG) and Primer 2 (SEQ ID NO:4:GCACTAGTTGGACTAGTGTTGTACTCA), above-mentioned primer is for the identification of the genetic sex of Cynoglossus semilaevis, the amplified production that it is characterized in that Cynoglossus semilaevis heredity female individuals is 2 dna fragmentations, and size is respectively 300-304 and 272-276bp; The amplified production of heredity male is 1 dna fragmentation, and size is 272-276bp.
Can also adopt other the female specific AFLP mark of Cynoglossus semilaevis or gunther sex-linked microsatellite marker primer for detection of the primer of Cynoglossus semilaevis genetic sex.
The normal milter of high-quality of adopting above-mentioned gunther sex-linked molecular marker screening to go out can be used for seed and breed in a large number, improves the physiology raun ratio of seed, also can be used for family and make up, and screens high female family.
2) artificial propagation and family are set up:
To step 1) in the male parent population that filters out and reach sexually matured female parent population and carry out temperature control control light and artificial induced spawning, the ovum of the seminal fluid of 1 tail milter and 1 tail raun is carried out artificial insemination sets up a family, and the family of setting up is carried out seed selection;
The number that carries out the family of seed selection is 30-50;
Adopt the belly extrusion to obtain the seminal fluid of milter and the mature egg of raun; To be placed in the tank of 1 2.5-3.5 cubic meter from the 50-100ml of family come-up fertilized egg and hatch and fry rearing, water temperature is controlled at 19-24 ℃;
3) detection of the cultivation of different family fries and female-male proportion:
With step 2) the fry of different familys use respectively different fluorochrome labels after, cultivate at same breeding water body, in the time of mixed breed 12-16 month, carry out body length, measured body weight and female, male other evaluation of physiological, with physiology raun ratio be higher than 45% and the family of fast growth be defined as high female, Fast Growth family, these Genealogy screenings are out proceeded to cultivate;
4) cultivation and the breeding of high female, Fast Growth family
To cultivate to match after the maturation from female, the milter of the female family of same height and raise up seed, or the milter of the raun of a high female family or milter and another high female family or raun matched raise up seed, can produce in a large number physiology raun ratio reaches more than 45% and the fast high female fry that grows, thereby improve the physiology raun ratio in the cynoglossus semilaevis cultivation seed, improve cynoglossus semilaevis cultivation output.
The present invention screens 1 of the chain microsatellite marker of Cynoglossus semilaevis sex chromosome, adopt this mark to set up the method for Cynoglossus semilaevis heredity raun and the evaluation of hereditary milter, set up the tongue sole family that family selective breeding goes out high female ratio by normal milter, thereby set up the method that improves physiology raun ratio in the cynoglossus semilaevis cultivation seed.Method of the present invention has the advantages such as accurate, efficient; physiology milter ratio too high (usually up to 70-90%) in the ubiquitous cultured population, physiology raun ratio is on the low side (being generally 10-30%) problem have been solved in the cynoglossus semilaevis cultivation industry; large-scale production and cultivation for semi-smooth tongue sole offspring breed; improve cultured output and economic benefit; promote the lasting, fast-developing of cynoglossus semilaevis cultivation industry, significant and using value.
Description of drawings:
Fig. 1: the chain microsatellite marker of sex chromosome is to the qualification result electrophoretogram of Cynoglossus semilaevis raun and milter.
Embodiment
The present invention is further detailed explanation below in conjunction with drawings and Examples.
One, the screening of the male parent population of Cynoglossus semilaevis high-quality
1, the acquisition of the chain microsatellite marker of Cynoglossus semilaevis sex chromosome:
The chain microsatellite marker sequence of sex chromosome that the present invention uses derives from the gene order-checking project that we carry out Cynoglossus semilaevis.Adopt the SOLEXA sequencing technologies to carry out respectively genome sequencing to Cynoglossus semilaevis female individuals and male, female individuals genome sequence and male genome sequence are compared, therefrom filter out 1 special microsatellite sequence of sex chromosome, called after SChen-SSR2, its male and female consensus sequence is SEQ ID NO:1, and female characteristic sequences is SEQ ID NO:2.
Male and female have fragment sequence (274bp): SEQ ID NO:1
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGGCCTATTTGTAATATTTCAGTACTAGATGTATCCCTACAAATTGTGTTTAGAGGTAAGCATTTAAATAGTGGTTTTTATTGACAAATATGCAGATTGGATGTCGGGAGGTCGTCGTGCAGCTCATATAACTAGACATTATCGCCTGTTGAGTTTGTGGCTAGAAGCCAACTGGAAGGTTAGATTGTAGAAAATTGTTTTTTTTTTTTTTTGTCGTG
TGAGTACAACAC TAGTCCAACTAGTGC
Female peculiar fragment sequence (302bp): SEQ ID NO:2
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGACAATTTTGTAATATTTCAATCCTAGATGTATCCCTATAAATTGTGTTTAGAGGTAAGCATTTAGATGTGGTTTAGAAGTGGTTTTTATTGTATTGTATTGTATTGTATTGTATTGTATGCAGCTTAGCTGTCAGGAGGTCGTCGTGTGGTTCATTTAACTAGACATTATCACCTGCTGAGTTTGTGGCCAGAAGCCAACTGGAAGGTTAGATTGTAGAAAACAGTTTATTTTTATGTGTTGTG
TGAGTACAACACTAGTCCAACTAGTGC
According to 1 pair of the primers of the chain microsatellite marker SChen-SSR2 of this sex chromosome, be respectively Primer 1:(SEQ ID NO:3:CAG TGTGT GTTTATTTAAAGGTTAGTTGGGG) and Primer 2 (SEQ ID NO:4:GCACTAGTTGGACTAGTGTTGTACTCA).
2, the checking of the chain microsatellite marker of sex chromosome: adopt 12 tail Cynoglossus semilaevis males and 12 tail female individuals DNA samples that the special microsatellite marker of this sex is carried out pcr amplification, 15 μ l systems, wherein distilled water 10.8 μ l are adopted in amplification; 10 * Buffer, 1.5 μ l; DNTP (2.5mM) 0.8 μ l; Each 0.4 μ l of upstream and downstream primer; For guaranteeing active last Takara enzyme (rTaq 5U/ μ l) the 0.1 μ l that adds of enzyme; Then fully spiral mixing packing adds corresponding dna profiling 1.5 μ l; 15 μ l altogether.The PCR response procedures is: 95 ℃ of 5min, 1 circulation; 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 1 circulation of 72 ℃ of 7min; After the PCR EP (end of program) in 4 ℃ of preservations.Amplified production is electrophoresis in 6% denaturing polyacrylamide gel, and dyes by silver staining method, then observes expansion product number of fragments and the size of microsatellite marker in the male and female individuality.The result shows that this microsatellite marker all amplifies 2 dna fragmentations in 12 tail rauns, and size is respectively 300-304 and 272-276bp; The amplified production of 12 tails heredity male is 1 dna fragmentation, and size is 272-276bp (Fig. 1).
3, the screening of the male parent population of high-quality
Adopt the special primer of cynoglossus semilaevis gunther sex-linked microsatellite marker SChen-SSR2 to identify the genetic sex of the male parent population of Cynoglossus semilaevis, the amplified production of Cynoglossus semilaevis heredity female individuals is 2 dna fragmentations, and size is respectively 300-304bp and 272-276bp; And the amplified production of hereditary male is 1 dna fragmentation, and size is 272-276bp.If certain bar physiology milter amplifies two DNA bands of 300-304 and 272-276bp, illustrate that this fish is pseudo-milter, i.e. heredity is upper for being male milter on female, the physiology.And only amplify the dna fragmentation of 1 272-276bp, then be normal milter.
Can also adopt other Cynoglossus semilaevis special numerator mark, (having delivered document according to the inventor carries out: Chen etc. such as the female specific AFLP mark of Cynoglossus semilaevis CseF382,2007, Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis) .Mar Biotech, 9:273-280.) or gunther sex-linked microsatellite marker primer CseF-SSR1 (having delivered document according to the inventor carries out: Chen et al., 2012 Induction of mito-gynogenetic diploids and identification of WW super-female using sex-specific SSR markers in half-smooth tongue sole (Cynoglossus semilaevis).Mar Biotechnol, 14:120-128.), Cynoglossus semilaevis breeding is carried out genetic sex with male parent population identifies, with genetic sex be male, simultaneously the physiology sex also be male parent fish selection out, be used for family structure and high female excellent strain seed selection.
Sequence and the primer sequence of CseF382 AFLP mark are:
Primer?CseF382N1
ATTCACTGACCCCTGAGAGCGGCGAAGTTAGGCAGTTCGCTGAGTCCAGTGCAAACATCAACGTTCTTATATGCCAACAGACTTTAGAAGCCAAGAATTATGTGTCATATTTGTTTTGACTCCAGGTTCAGTTTACTTAGGACAAACTCAGGGATTTGAAATGACGAGCAACATCTTATCGCCTCAAGTGCACATGGGTAAAAAGATCCTTCCGAGCCAGGGCTGGAGAAAGCAGGCTTCTCAGGTGACACAGTGGGTTTGGCCGAGACTAGTTTTAC
AATGATGGTGCCACAAAAAAAGAAAGGGAAACTCTATGAGTGGACACAGACTGAG
ACATTTGTCGTGTGTGAG
Primer?CseF382C1。
The sequence of CseF-SSR1 microsatellite marker is:
A: female peculiar microsatellite marker sequence (218bp)
Gaggccgacaggatcgtacataatcccaacttcacaataactccacaattggcactttttgttttgtttttctcttttactttcttaacaattatacacactcggagcccgtatcgcaatgtcacaccgagggtttgtttcttctaaggtcacacacacacacacacacacaggcatgactgaagagtttctgtcg
aaaaccaccggagtacgtcgta
B: male and female have microsatellite marker sequence (206bp)
Gaggccgacaggatcgtacatcctcccaacttcacaataactccacaattggcacttttttttttctcttttactttcttaacaattacacacactcggagcccgtatcacaatgtcacaccgagggtctgtttcttctaaggtcaaacacacacacacgggcatgactgaagagtttcagttg
aaaaccaccggagtacgtcgta。
We carried out pseudo-milter detection in Yantai Huanghai Aquatic Products Co.,Ltd., Haiyang City and Mingbo Aquatic Product Co., Ltd., Laizhou to stock male parent population in recent years to adopt said method, now testing result were summarized in table 1.
Table 1:2011-2012 detects summary sheet in 2 companies in Haiyang and Laizhou to male parent population
By as seen from Table 1, in the male parent population of the Cynoglossus semilaevis ordinary group of propagating artificially in the cynoglossus semilaevis cultivation field, nearly 27.8% milter is pseudo-milter, and the ratio of normal milter is 72.2%, shows that thus having an appointment in the common seed of Cynoglossus semilaevis artificial propagation 28% is the offspring of pseudo-milter.
4. the comparison of normal milter offspring and pseudo-milter offspring seed raun ratio
In recent years, genetic sex and the physiology sex ratio of normal milter (being the ZZ type in the heredity) colony and pseudo-milter (being to be milter on ZW type, the physiology in the heredity) offspring of colony seed compared in our investigation, the result shows that the ratio of hereditary raun among the normal milter offspring of colony (being the ZW type in the heredity) is 47%-51%, and the ratio of physiology raun (gonad development is ovary) is 41%-43%; On the contrary, hereditary raun (being the ZW type in the heredity) ratio in the pseudo-milter offspring of the colony seed is up to 69%-71%, but the ratio of physiology raun (gonad development is ovary) only has 5.2%-6.2%, is physiology milter (table 2) and the hereditary raun sex reversal of 91.2-92.5% is arranged.And physiology milter poor growth, individual little does not have commercial value.Therefore, pseudo-milter need to be removed from milter colony in the Cynoglossus semilaevis artificial propagation with in growing seedlings, and normal high-quality milter is picked out for seed breeding, physiology raun ratio in the guarantee tongue sole seed solves the problems such as the milter ratio is too high in the common cultured population, growth rate is slow, cultured output is low like this.
The normal milter of table 2 and the pseudo-milter offspring of colony seed sex ratio are relatively
The normal milter of adopting said method to filter out can be used for raising up seed seed in a large number, the physiology raun ratio in the cultured population seed can be brought up to about 40%, can apply.And for more a high proportion of raun, just need to carry out family selective breeding and obtain high female family.
Two, artificial propagation and family are set up
The above-mentioned physiology raun ratio of the physiology raun ratio among the normal milter offspring in the pseudo-milter offspring that studies show that has application value.But, the above results is that the normal milter seminal fluid of many tails mixes the result that insemination obtains.So, whether variant on physiology raun ratio between the different normal milter offsprings? can cultivate the higher seed of physiology raun ratio among the offspring? therefore, we adopt normal milter parent population to carry out family structure and high female Genealogy screening.We are to the male parent population of normal high-quality that filters out and reach sexually matured 3 ages and above female parent population carries out temperature control control light and artificial propagation, carry out parent fish pond water temperature and illumination control in front about 2 months in breeding; Water temperature by per 10 days rising 0.5-1 ℃, progressively be elevated to 22-23 ℃ from 17-19 ℃, and maintain 22-23 ℃; Illumination then prolonged 1 hour by per 10 days, by 8 hours every days light application time extend to illumination 16 hours every days, and maintain illumination in 16 hours.Touch inspection by observation and hand and select sexual gland obviously to swell, gently press sexual gland to have obvious sense of fulfillment and sexual gland front end that the raun of certain pliability is arranged in both sides up and down, be used for artificial induced spawning and breed; Male parent population is then selected gently to squeeze the sexual gland position, sees that seminal fluid outflow person uses.Adopt the belly extrusion to obtain the seminal fluid of milter and the mature egg of raun: after raun picks up, pin the gonopore position with towel first from water, prevent that ovum from flowing out, wrap the fish body with towel rapidly simultaneously, gonopore one side is exposed at the outside and wipes seawater.Push gently from back to front the sexual gland position with hand in both sides up and down, ovum is extruded, be connected in the dry beaker.During semen collection, push ripe milter belly with hand, with suction pipe the seminal fluid of extruding is forwarded in the dry bottle and preserve, or directly seminal fluid is extruded in the beaker of ovum.Smart ovum is mixed, add 2 times of seawater to the ovum amount and namely finish the insemination operation, the unnecessary seminal fluid of flush away can change in the hatching cylinder and hatch behind the 20min.The seminal fluid of 1 tail milter and the ovum of 1 tail raun are carried out artificial insemination, namely form a family, will be placed in the tank of 1 2.5-3.5 cubic meter from the 50-100ml of family come-up fertilized egg and hatch and fry rearing, water temperature is controlled at 19-24C; Should set up family 30-50, each family should be cultivated 300-500 tail fry and be used for the screening of high female family and growth fraction at every turn.
2010, we built together in Mingbo Aquatic Product Co., Ltd., Laizhou and have found 35 of tongue sole familys, and wherein cultivating the family that survives is 25, and existing information with 25 familys is summarized in table 3.
Table 3:2010 tongue sole families is set up situation (20100701-20110410)
Three, the detection of the cultivation of different family fries and female-male proportion:
When different family fry growths during to the 12-16 centimeter length, each family is chosen at random 200-250 tail fry and is adopted fluorescent dye to carry out mark, the identical dye combinations of same family mark, different family fries behind the mark are placed on carry out mixed breed in the same cement pit, every square meter water body is put 30-40 tail fry in a suitable place to breed, when fry growth during to the 20-25 centimeter length, cultivation density is adjusted into every square meter water body 15-20 tail fry, continuing cultivation raun (large specification fish) extremely wherein grows to more than 40 centimetres, when body weight 350 grams are above, the physiology sex identification and the body that carry out different family fishes are long, measured body weight, physiology sex identification adopt the method at strong illumination sexual gland position to carry out.With fishing net fish is got, from the irradiation of sexual gland position, the fish body back side, observe the sexual gland CF with strong fluorescent lamp in outside of belly sexual gland position, if sexual gland is longer and narrower, color is faint yellow or transparence is raun, if sexual gland is short and little, and color is black or navy blue then is milter.Reach physiology raun ratio more than 45% and the family of fast growth is defined as high female family, these Genealogy screenings are out proceeded to cultivate.
Transfer to Changyi tongue sole plant after we respectively select 200 tail fries to carry out fluorescence labeling in June, 2011 and carry out mixed breed test from 22 family, altogether mark 4323 tail fries transfer to three new aquatic products Co., Ltds, be placed in 2 culturing pools and cultivate.Carry out body length, measured body weight every 3-5 month tongue sole family fry to mixed breed subsequently, when experiment finishes, choose several familys and carry out the physiology sex identification.Now different family growth fractions be the results are summarized in table 4, with the normal milter family of representativeness physiological
Other qualification result is summarized in table 5.
Table 4:2010 tongue sole families growth fraction
Physiology raun ratio summary sheet in the representative normal milter family of table 5:2010 Cynoglossus semilaevis
By as seen from Table 5, normal milter is individual different, physiology raun ratio among its offspring also is not quite similar, although the physiology raun ratio (19%-68%) in the normal milter offspring seed is all apparently higher than pseudo-milter offspring (referring to table 1), but the physiology raun proportional difference between the different familys of normal milter is also larger, such as, the physiology raun ratio of No. 16 familys is up to 68%, next is No. 57 familys, its physiology raun ratio also reaches 47%, apparently higher than other family, so these 2 familys are high female family; And the physiology raun ratio of No. 44 familys only has 19%, therefore, can filter out the high family of physiology raun ratio by the family selective breeding mode.By being compared, the proterties such as the growth of 25 familys, sex ratio just filter out that 1 physiology raun ratio is high, superior families-No. 16 family of fast growth among the present invention, the cultivation survival rate of this family is up to 73%, and its relative weight gain rate during cultivation in 300 days is up to 8.1; Also screen simultaneously high, faster family-No. 57 family of growing also of 1 raun ratio, its physiology raun ratio reaches 47%, and its relative weight gain is also higher, reaches 4.1.In addition, the cultivation survival rate of No. 17 familys is also up to 75%, and is suitable with No. 16 familys, and the daily gain of No. 17 familys also has 0.45, and these familys all are the superior families with better proterties.Therefore, we filter out several female ratios high, grow fast or the high superior families (No. 16, No. 57 and No. 17) of survival rate to adopt the technology of the present invention, and these familys all have great using value and wide promotion prospect.
Four, the cultivation of high female superior families and breeding
Be that high female family adult fish more than 45% is cultivated as reserve parent fish with the physiology raun ratio that screens, the high female family reserve parent fish of difference carried out being blended in the same cement pit behind the fluorescence labeling cultivate, when arrive mating season, carry out temperature control control light, when the parent population gonadal maturation, carry out artificial induced spawning and breeding.During artificial propagation, to cultivate to match after the maturation from female, the milter of the female family of same height and raise up seed, or the milter of the raun of a high female family or milter and another high female family or raun matched raise up seed, can produce in a large number so the high female seed of physiology raun ratio height, fast growth, thereby improve the raun ratio in the cynoglossus semilaevis cultivation colony, improve cultured output and the economic benefit of Cynoglossus semilaevis.Existing cultivation result shows that female ratio has significant raising than the family that does not have screening among the high female family offspring that we set up, and has good application value.
Claims (4)
1. a method that improves cynoglossus semilaevis cultivation seed raun ratio comprises the steps:
1) screening of the male parent population of Cynoglossus semilaevis: the male parent population of Cynoglossus semilaevis is carried out genetic sex identifies, with genetic sex be male, simultaneously the physiology sex also be male parent fish selection out;
2) artificial propagation and family are set up:
To the male parent population that filters out in the step 1) with reach sexually matured female parent population and carry out temperature control control light and artificial induced spawning, the ovum of the seminal fluid of 1 tail milter and 1 tail raun is carried out artificial insemination set up a family, the family of setting up is carried out seed selection;
3) detection of the cultivation of different family fries and female-male proportion:
With step 2) different family fries use respectively different fluorochrome labels after, cultivate at same breeding water body, in the time of mixed breed 12-16 month, carry out body length, measured body weight and female, male other evaluation of physiological, with physiology raun ratio greater than 45% and the family of fast growth be defined as high female family, these Genealogy screenings are out proceeded to cultivate;
4) cultivation and the breeding of high female, Fast Growth family
To cultivate to match after the maturation from female, the milter of the female family of same height and raise up seed, or the milter of the raun of a high female family or milter and another high female family or raun matched raise up seed, high and the fast high female fry that grows of a large amount of production physiology raun ratios, thereby the physiology raun ratio in the raising cynoglossus semilaevis cultivation seed;
The male parent population of Cynoglossus semilaevis is carried out genetic sex identify to be to increase by the primer that designs from the cynoglossus semilaevis gunther sex-linked molecular labeling and to identify in the described step 1); Wherein the cynoglossus semilaevis gunther sex-linked molecular labeling is the chain microsatellite marker SChen-SSR2 of Cynoglossus semilaevis sex chromosome, is divided into the total fragment sequence of male and female and female peculiar fragment sequence;
The total fragment sequence of male and female is SEQ ID NO:1:
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGGCCTATTTGTAATATTTCAGTACTAGATGTATCCCTACAAATTGTGTTTAGAGGTAAGCATTTAAATAGTGGTTTTTATTGACAAATATGCAGATTGGATGTCGGGAGGTCGTCGTGCAGCTCATATAACTAGACATTATCGCCTGTTGAGTTTGTGGCTAGAAGCCAACTGGAAGGTTAGATTGTAGAAAATTGTTTTTTTTTTTTTTTGTCGTGTGAGTACAACACTAGTCCAACTAGTGC；
Female peculiar fragment sequence is SEQ ID NO:2:
CAGTGTGTGTTTATTTAAAGGTTAGTTGGGGACAATTTTGTAATATTTCAATCCTAGATGTATCCCTATAAATTGTGTTTAGAGGTAAGCATTTAGATGTGGTTTAGAAGTGGTTTTTATTGTATTGTATTGTATTGTATTGTATTGTATGCAGCTTAGCTGTCAGGAGGTCGTCGTGTGGTTCATTTAACTAGACATTATCACCTGCTGAGTTTGTGGCCAGAAGCCAACTGGAAGGTTAGATTGTAGAAAACAGTTTATTTTTATGTGTTGTGTGAGTACAACACTAGTCCAACTAGTGC。
2. the method for claim 1 is characterized in that described microsatellite marker SChen-SSR2 is used for the design primer, and designed primer is Primer 1 and Primer 2, and wherein Primer 1 primer sequence is SEQ ID NO 3:
CAG?TGTGT?GTTTATTTAAAGGTTAGTTGGGG；
The primer sequence of Primer 2 is SEQ ID NO 4:
GCACTAGTTGGACTAGTGTTGTACTCA。
3. method as claimed in claim 2 is characterized in that described primer Primer 1 and Primer 2 are 2 dna fragmentations at the amplified production of Cynoglossus semilaevis heredity female individuals, and size is respectively 300-304 and 272-276bp; Be 1 dna fragmentation at hereditary male amplified production, size is 272-276bp.
4. the method for claim 1 is characterized in that described step 2) artificial propagation and family foundation, the number that wherein carries out the family of seed selection is 30-50.
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| CN102134593A (en) * | 2010-12-23 | 2011-07-27 | 中国水产科学研究院黄海水产研究所 | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis |
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| CN102134593A (en) * | 2010-12-23 | 2011-07-27 | 中国水产科学研究院黄海水产研究所 | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis |
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| 木云雷 等.半滑舌鳎亲鱼培育技术研究.《水产科学》.2007,第26卷(第10期),535-538. |
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