CN103814810B - Method of obtaining oyster distant intergeneric hybrid - Google Patents
Method of obtaining oyster distant intergeneric hybrid Download PDFInfo
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- CN103814810B CN103814810B CN201410072688.4A CN201410072688A CN103814810B CN 103814810 B CN103814810 B CN 103814810B CN 201410072688 A CN201410072688 A CN 201410072688A CN 103814810 B CN103814810 B CN 103814810B
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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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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a method of obtaining an oyster distant intergeneric hybrid. The method includes a step of collecting different intergeneric parents, labeling each identified species and synchronously ripening; a step of, after the gonads of the parents are ripe, dissecting sperms and eggs, isolating strictly and performing intergeneric hybridization and matching to obtain a selfing group and a hybridization filial generation group of two parents, and a step of cultivating the filial generation to obtain the survival distant intergeneric hybrid. At the same time, the type of the hybrid is determined by performing heredity identification by utilization of molecule and adoption of cytology means. According to the method, oyster intergeneric hybridization is performed for the first time and the survival intergeneric hybrid is obtained, thus providing a rare scarce material for analysis of oyster genetic differentiation, reproductive isolation and species formation and developing a novel route for genetic breeding of the oyster. The method has advantages of simple operation, high feasibility, and the like.
Description
Technical field:
The invention belongs to shellfish Biotechnology in Genetic Breeding field in modern marine agricultural, be specifically related to a kind of method obtaining distant hybrid between oyster genus.
Background technology:
Intergeneric cross be equal in do not belong to hybridization between interior species together, it can expand vegeto-animal gene pool significantly, exchanges, introduce xenogenesis beneficial gene, create unprecedented variation type, even can synthesize new species (Lou Yundong, 1999) between promoting kind.
For aquatic livestock, intergeneric cross mainly concentrates on fish breeding, relatively less to the research of shellfish.Wherein, be relatively successfully chlamys farreri and Patinopecten yessoensis distant hybridization (Yang Aiguo etc., 2003), obtain the hybridization group with growth vigor, achieve ideal economic benefit.For oyster, although the Ostreidae found in China has 5, namely huge oyster belongs to (Crassostrea), oyster belongs to (Ostrea), little oyster belongs to (Saccostrea), tooth oyster belongs to (Dendostrea), hyoid bone oyster belongs to (Parahyotissa) etc. (summer builds up the Army, 2008), but there is not yet intergeneric cross report, what relative reporter was more is interspecific cross research.As far back as 1882, Bouchon-Brandely just carried out the interspecific cross (Bouchon-Brandely, 1882) of Portuguese oyster (Ostreaangulata) and flat oyster (O.edulis).In nineteen fifty, Davis have studied the interspecific cross of Pacific oyster O.gigas × American oyster O.virginica and American oyster O.virginica × Olympia oyster O.lurida.
It is worth mentioning that, American scholar Gaffney & Allen summed up the interspecific cross that huge oyster belongs to oyster in 1993, propose, oyster interspecific cross is before this owing to not yet carrying out Genetic identification, only observed by phenotypic character, think and hybridize successfully, its confidence level height is open to suspicion.What is more important, they propose species hybrid in huge oyster genus and can not survive, and this just causes in 15 years approximately after this does not almost have oyster interspecific cross to study report.The Institute of Oceanology of the Chinese Academy of Sciences, Chinese Academy of Sciences's South Sea ocean research in one's power Chinese Marine University under state key combines fund on fund, national face, funded projects such as country 973 etc., complete the interspecific cross that huge oyster belongs to oyster, obtain real species hybrid, resolve huge oyster to belong to oyster interspecific cross mechanism and (permitted to fly, 2009; Zhang Yuehuan, 2012; Xuetal, 2009; Zhangetal, 2012; Huoetal, 2003).So, on interspecific cross basis, can hybridize between belonging in Ostreidae, edge advantage far away can be obtained, whether can obtain survival filial generation, thus to be formed for oyster species and genetic evolution provides precious intermediate materials to be innovative point place of the present invention.
Summary of the invention:
In order to attempt the feasibility of oyster inter-genera distant hybridization, obtaining oyster distant hybrid intermediate materials, opening up an oyster new technology of breeding, the invention provides a kind of distant hybridization method obtaining oyster and bigener.
The distant hybridization method that acquisition oyster of the present invention bigeners, is characterized in that, comprise the following steps:
A, parent gather: gather Ostreidae and do not belong to interior kind together as parent; Because oyster shell form is very large along with environmental change, being difficult to distinguish in appearance from it is any oyster, therefore on Phenotypic Observation basis, also will carry out species identification by means of Molecular tools;
B, synchronous accelerating: carry out mark to each kind, carry out gonad maturity accelerating in indoor, guarantee the synchronism of gonad maturity; Be directed to the ecological habit of each oyster, carry out the regulation and control of the ecological factor such as temperature, salinity, guarantee the synchronism of gonad maturity.
C, take smart ovum: after parent's gonad maturity, dissect smart ovum, be strictly separated, carry out the intergeneric cross pairing of species between two, obtain selfing group and the filial generation of reciprocal cross group thereof of two parents; To the parent used, immobilization material, carry out subsequent analysis.Intergeneric cross due to oyster often there will be gamogony sovereignty nuisance in various degree, is directed to different combinations of pairs, obtains unidirectional fertilization group or all nonfertilization is all normal.
D, filial generation are cultivated: after obtaining selfing group and hybridization group, carry out filial generation cultivation, in the larvae cultivation stage, intergeneric cross D shape larva may occur that delay is ingested situation, cultivate through 3-7d, can normally ingest, obtain hybridization juvenile mollusk through larvae cultivation, juvenile mollusk is through Transitional culture and form acquisition and bigener;
E, Genetic identification: because in fertilization, larvae cultivation process, be easy to occur contamination phenomenon, but also may along with the generation of natural androgenesis, gynogenesis, polyploid phenomenon.So be difficult to ensure whether hybrid is hybrid generation.In fertilization and larvae cultivation process, adopt molecular labeling to confirm bigener hybrid generation or there occurs thelykaryon or androgenesis phenomenon, and utilize flow cytometer to carry out Ploidy detection to it, whether have natural polyploid phenomenon to occur
Compared with the present invention, Yang Aiguo etc. disclosed the documents 1 that Authorization Notice No. is CN1283143C in 2003, a kind of high yield chlamys farreri and Patinopecten yessoensis distant hybrid breeding method (Yang Aiguo etc., 2003).So, the present invention, compared with documents 1, also exists following difference.The first, research purpose of the present invention is different from documents 1, documents 1 emphasis obtains high yield hybrid, only improve culture trait, moreover, before very early, Chinese scholar (old fur coat etc., 1993) has just carried out the research of variety classes scallop intermolecular hybrid, illustrate that scallop distant hybridization feasibility predicts in advance, hybrid vigour is also have report in advance; And emphasis of the present invention obtains survival filial generation, provide intermediate materials for oyster genetic evolution and species thereof are formed, simultaneously for edge back cross breeding far away and multiple cross thereof lay the foundation from now on.The second, the technology of the present invention method is different from documents 1, documents 1 is scallop breeding method, and the present invention is oyster Cultivating techniques, the reproductive biology of two class economic shellfishes and fry production process thereof also exist larger difference, accumulated temperature as scallop is relatively low, and the accumulated temperature of oyster is relatively high; Scallop is ejaculation of laying eggs, and oyster takes smart ovum; Scallop cultivation temperature is lower, and oyster cultivation temperature is higher; Scallop is collected seedling by polyethylene mesh sheet, and oyster is collected seedling with shell; Scallop juvenile mollusk is that mesh bag is bred as, and young shellfish scallop is raised in cages into; And oyster marine be bred as and form.Three, the present invention is different from documents 1 on filial generation identification requirement, as long as documents 1 only thinks hybridization, its filial generation is exactly hybrid, ignore the phenomenons such as in crossover process sperm pollution, larva pollution, spontaneous gynogenesis, androgenesis, natural polyploid to hybridizing the impact brought, whether hybrid is that real both sexes blendling makes us querying; And the present invention adopts Molecular Identification, flow cytomery ploidy, on traditional phenotype identification basis, modern molecular and cytology means are adopted to ensure that the reliability of bigenering.Four, the scope of application of the present invention is different from documents 1, and documents 1 method is only only applicable to the distant hybridization of two kinds of scallops, and the present invention is applicable to the intergeneric cross of all oysters.
In a word, the present invention is capturing on oyster interspecific cross basis, is groping first to the one of oyster intergeneric cross, is also the one challenge to cultivating saying of bigenering traditionally; For preparation distant hybridization material, the genetic evolution of research oyster, species formation etc. provide the foundation; Also for oyster distant hybridization breeding provides a new technology.
The oyster that the present invention obtains survival first bigeners, for oyster genetic variation and genetic differentiation, reproduction isolation and species thereof form analysis and obtain rare rare material, also for the genetic breeding of oyster opens a new road.
Accompanying drawing illustrates:
Fig. 1 is Technology Roadmap of the present invention.
Fig. 2 is parents and filial generation thereof.A: Portuguese oyster parent; B: Ostrea cucullata parent; C: hybrid generation.
Fig. 3 is progeny inherit qualification figure (ITS2).Note: M is 2000bpDNAmarker; A, Portuguese oyster parent; AC, Portuguese oyster ♀ × Ostrea cucullata ♂ filial generation; C, Ostrea cucullata parent.
Fig. 4 is the filial generation ploidy result of flow cytometry.A represents that parents are 2 times of bodies; B represents that hybrid generation is also still 2 times of bodies.
Embodiment:
With the inter-genera distant hybridization that oyster huge in Ostreidae belongs to and little oyster belongs to for embodiment further illustrates the present invention, but be not construed as limiting the invention.
Embodiment 1:
A, parent gather: at the beginning of 2013 4 months, according to technology path Fig. 1, gush collect huge oyster and belong to Portuguese oyster at Shen Zhendong, also have a kind of shell inner edge to have the oyster of a lot of little tooth, through qualification, are the Ostrea cucullatas (Fig. 2) that little oyster belongs to.This two seed oyster from it in appearance, does not see any difference, but after opening shell, can distinguish Ostrea cucullata, and Portuguese oyster also needs Molecular Identification.By compound COI Molecular Identification, Portuguese oyster can produce 222bp specific fragment (Fig. 3, WangH, GuoX. (2008) IdentificationofCrassostreaariakensisandrelatedoystersby multiplex species-specificPCR.JShellfishRes27:481 – 487).For parent, be first numbered, carry out living body sampling with reference to auspicious extra large & Wang Zhao duckweed invention disclosed patented technology (Authorization Notice No.: CN101849520B) afterwards, the gill filament that takes a morsel, as sample, carries out molecular recognition after alcohol fixation.
B, synchronous accelerating: in mid-April, 2013,200 individualities are respectively screened as parent to this two seed oysters Portuguese oyster differentiated and Ostrea cucullata, be positioned in Shenzhen Long Keyuan choice seafood aquiculture Co., Ltd cement pit and gonad maturity accelerating carried out to this two seed oyster, micro-air-charging incubation.Throw something and feed every day chlorella, Nitzschia closterium minutissima, yeast, feeding volume, using micro-excessive in standard, ensures that parent has enough bait.During accelerating, water temperature is at 23.2-26.5 DEG C, and salinity is at 28-30ppt.Because this two seed oyster is often living together, belong to congruence ideal, so accelerating condition is more consistent.Guarantee the synchronism of gonad maturity.
C, take smart ovum: in mid-May, 2013, two seed oyster sexual glands are synchronously ripe, take the mode of dissecting gamete, obtain monomer essence ovum.Obtain ovum method, utilize 300 mesh sieve tulles to filter out tissue block, Recycle of filtrate 500 order washes ovum, collects the ovum on 500 mesh sieve tulles, soaks 30-60min afterwards as in 3L Plastic Drum, becomes circle into standard with ovum.Sperm acquisition methods, about 10min before ovum becomes circle, obtains monomer sperm, utilizes 500 mesh sieve tulles to filter, and removal impurity, collects filtered fluid, through sea-water activated 5-10min, be placed in 100ml beaker, sperm can acutely be beated in the seawater.To the smart ovum obtained, strictly isolate.Carry out the pairing between two between monomer afterwards, form Portuguese oyster, Ostrea cucullata 2 selfing groups, Portuguese oyster ♀ × Ostrea cucullata ♂, Ostrea cucullata ♀ × Portuguese oyster ♂ 2 hybridization group, has made 9 combinations altogether.From Fertility levels, Portuguese oyster ♀ × Ostrea cucullata ♂ group has higher Fertility levels >=90%, and Ostrea cucullata ♀ × Portuguese oyster ♂ fertilization rate is 0, namely has unidirectional fertilization phenomenon.
D, filial generation are cultivated: Portuguese oyster ♀ × Ostrea cucullata ♂ group obtains a large amount of larva, and front 5d does not ingest, but after this, normal growth, abnormal, obtain some juvenile mollusks.Compared with two parent's groups, Portuguese oyster ♀ × Ostrea cucullata ♂ does not have growth, survival advantage between group larval phase, but can complete metamorphosis, is converted into juvenile mollusk, afterwards normal growth, has growth potential.In 9 groups, 5 groups are only had to obtain hybridization juvenile mollusk.Be bred as through juvenile mollusk and form to obtain afterwards and bigener.
E, Genetic identification: because in fertilization, larvae cultivation process, be easy to occur contamination phenomenon, but also may along with the generation of natural androgenesis, gynogenesis, polyploid phenomenon.So be difficult to ensure whether hybrid is hybrid generation.For this reason, adopt molecular labeling can confirm that hybrid is hybrid generation (WangY, GuoX. (2008) ITSlengthpolymorphismin oystersanditsuseinspeciesidentification.JShellfishRes27: 489 – 493), still there occurs thelykaryon or androgenesis phenomenon.Flow cytometer also to be utilized to carry out Ploidy detection to it, whether have natural polyploid phenomenon to occur.Discovery when carrying out Genetic identification for hybrid generation, Ostrea cucullata ♀ × Portuguese oyster ♂ larva is both sexes blendling truly, is really bigener (Fig. 3).Found by flow cytomery, hybrid is dliploid, not yet occurs natural polyploid phenomenon (Fig. 4).
Claims (1)
1. obtain the distant hybridization method that oyster bigeners, it is characterized in that, comprise the following steps:
A, parent gather: the Ostrea cucullata of the Portuguese oyster and little oyster genus that gather huge oyster genus is as parent;
B, synchronous accelerating: carry out mark to each kind, carry out gonad maturity accelerating in indoor, guarantee the synchronism of gonad maturity;
C, take smart ovum: after parent's gonad maturity, dissect smart ovum, be strictly separated, carry out the intergeneric cross pairing of species between two, obtain selfing group and the filial generation of reciprocal cross group thereof of two parents;
D, filial generation are cultivated: after obtaining selfing group and hybridization group, carry out filial generation cultivation, and obtain hybridization juvenile mollusk through larvae cultivation, juvenile mollusk is through Transitional culture and form acquisition and bigener;
E, Genetic identification: in fertilization and larvae cultivation process, molecular labeling is adopted to confirm bigener hybrid generation or there occurs thelykaryon or androgenesis phenomenon, and utilize flow cytometer to carry out Ploidy detection to it, whether there is natural polyploid phenomenon to occur, confirm times body of hybrid generation.
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