CN106069717A - A kind of method that hermaphroditism Thallus Porphyrae Double-haploid population is set up - Google Patents
A kind of method that hermaphroditism Thallus Porphyrae Double-haploid population is set up Download PDFInfo
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Abstract
The invention discloses the method that the Double-haploid population of a kind of hermaphroditism Thallus Porphyrae is set up；Take color distortion obvious hermaphroditism thallus of porphyra (gametocyte) as hybrid strain；The parents' thallus forming anthreid is mixed, collects carpospore；The carpospore single culture choosing tool germ tube becomes filamentous (sporinite)；Induce and obtain the conchospore of this filamentous, cultivate to macroscopic F1Thallus, the thallus such as more than 90% is the chimera containing parents' color, then be used for setting up Double-haploid population；Separate each color lump on four color lump chimeras, obtain monospore or the isolated Somatic Cell Culture extremely macroscopic thallus of each color lump, cultivated by single of this thallus and self-fertilization obtains the pure lines filamentous of each color lump on every four color lump chimeras, form Double-haploid population.The present invention solves the establishment problem of hermaphroditism Thallus Porphyrae Double-haploid population, and the structure for its genetic linkage maps lays the foundation.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the side that a kind of hermaphroditism Thallus Porphyrae Double-haploid population is set up
Method.
Background technology
Porphyra yezoensis Ueda (Pyropia yezoensis) in hermaphroditism Thallus Porphyrae is the large-scale food that a kind of nutritive value is the highest
With Sargassum, extensively cultivated in the North of Yangtze River area of China, the annual economic benefit creating about 5,000,000,000 yuan, Porphyra yezoensis Ueda simultaneously
Extensive cultivate transferable and fixing substantial amounts of carbon (preventing Ocean acidification) and the element such as nitrogen and phosphorus (preventing eutrophication),
To keeping Offshore Ecology balance, there is the most great meaning.The cultivation history of Porphyra yezoensis Ueda was up to for more than 40 years, was the most formed
Nursery, cultivate, process and the complete industrialization system such as sale, conversely, for fundamental researches such as its hereditism always
It is in the stage of relative deficiency, has not yet to see the relevant report of Porphyra yezoensis Ueda genetic linkage maps and physical map.Heredity is even
The structure of lock collection of illustrative plates is the important step in genome research, is gene mapping and clone, genome structure and functional study
Basis.At present, China has constructed the genetic linkage map of large quantities of crop, forest and medicinal plants, such as Oryza sativa L., Semen Tritici aestivi, Semen Maydis
Deng industrial crops, for people from molecular level awareness and understanding plant characteristic and growth-development law, carry out the something lost of important character
Pass and analyze, separate and clone important gene etc. and made major contribution.
The correlational study of Sargassum genetic linkage maps is started late, the most Thallus Laminariae (Thallus Eckloniae) (Laminaria japonica),
Porphyra haitanensis (Pyropia haitanensis), Thallus Laminariae (Undaria pinnatifida) and water cloud (Ectocarpus
Siliculosus) there is the report that genetic linkage maps builds in.
Difficult point during Porphyra yezoensis Ueda genetic map construction hybridizes exactly, because Porphyra yezoensis Ueda is hermaphroditic kind
Class, during hybridization, self-fertilization and allogamy are carried out simultaneously, and autogamous probability allosome to be far above is subject to
Essence, therefore difficulty is bigger when identifying the heterozygosis filamentous that allogamy is formed.
Summary of the invention
It is an object of the invention to provide a kind of method that hermaphroditism Thallus Porphyrae Double-haploid population is set up.
It is an object of the invention to be achieved through the following technical solutions:
The present invention relates to a kind of method that hermaphroditism Thallus Porphyrae Double-haploid population is set up, described method includes walking as follows
Rapid:
S1, choose thallus color distortion obvious hermaphroditism thallus of porphyra (gametocyte) as hybrid strain；
S2, choose formed anthreid parents' thallus mixed culture, to parents' thallus, begin with carpospore cyst
Formation time, parents' thallus is separated single culture, until each the carpospore cyst on thallus is reached maturity, collects respectively
Carpospore；
S3, when carpospore formed germ tube time, take the carpospore single culture of single sprouting so that it is sprout into a Dan Ke
Grand filamentous (sporinite) algae falls；
S4, cultivate described monoclonal filamentous algae and fall the conchospore that diffuses after induction, to macroscopic F1Lobate
Body, examines under a microscope F1Thallophytic color and color lump quantity (can randomly select the F of more than 10001Thallus), as
The F of more than 90%1Thallus is the chimeric thallus containing parents' color and color lump number is 2 pieces, 3 pieces or 4 pieces, then this monoclonal
Filamentous algae to fall be by the heterozygosis filamentous of biparent cross, its F1The chimeric thallus having four color lumps in Dai can be used for
Build Double-haploid population；
S5, from the chimeric thallus containing four color lumps, separate single color lump, take the list that this color lump diffuses after induction
Spore or isolated somatic cell, cultivate to macroscopic thallus, take single cultivation, and eventually through this, single thallus is autologous is subject to
Essence obtains every four color lumps and is fitted together to the pure lines filamentous of each color lump on thallus, thus sets up into Double-haploid population.
In the present invention, the thallus color for the parents of hybridization must differ greatly, and be with the naked eye easily discriminated.
Four color lump chimeras must be selected as the material of informative population, prevent from destroying the hereditary constitution of DH colony, cause serious inclined
Segregation phenomenon.The single color lump separated from chimeric thallus cannot be directly used to the structure of Double-haploid population, it is necessary to this
Thallus or the thallus of Somatic Embryogenesis that the monospore of color lump is sprouted are material, prevent this color lump from separating on chimera
It is possible to be fertilized with other color lump before getting off, causing the filamentous obtained is the filamentous of heterozygosis.
In above-mentioned steps S2, as being mixed to reaching maturity always, regather carpospore the most infeasible, because if mixed
Close and cultivate to reaching maturity, then the carpospore on parent a sticks on the thallus of parent b after likely discharging, and is so receiving
The carpospore of parent a may be mixed with the when of collecting the carpospore of parent b, cause the result of mistake；Because Porphyra yezoensis Ueda is female
Male consubstantiality, crossover process was both formed on each parent's thallus male gamete, also can form female gamete, say, that be every
Individual parent's thallus both can do male parent can also do female parent, so parents' thallus will be divided before diffusing carpospore
Opening, like this, the female parent of the carpospore that certain thallus diffuses is exactly this thallus, and another is then male parent.
Preferably, in step S2, the parents' thallus having formed anthreid obtains as follows: take parent's silk
The algae section of shape body is placed in temperature 25 ± 1 DEG C, photon flux density 20-30 μm ol m-2·s-1, under conditions of photoperiod 10L:14D
Quiescent culture, culture fluid is the sterilization sea water of interpolation 2% (v/v) MES nutritional solution, cultivates the nutrition algal filament growth to more than 50%
Maturation, forms conchosporangia；Take out half and contain the algal filament of conchosporangia in temperature 19 ± 1 DEG C, photon flux density 40-
50μmol m-2·s-1, to cultivate under conditions of photoperiod 10L:14D, culture fluid is the sterilization of interpolation 2% (v/v) MES nutritional solution
Sea water, in the most ripe conchosporangia release conchospore to culture fluid；By the culture fluid containing conchospore (by 200 mesh sieve thin,tough silk mistakes
Filter obtains conchospore suspension), the leaflet continuing to cultivate to this conchospore germination is grown up, and starts shape to thallus ending
Become anthreid,.
Preferably, in step S2, the cultivation temperature of described mixed culture 19 ± 1 DEG C, photon flux density 40-50 μm ol
m-2·s-1, photoperiod 10L:14D, culture fluid is the sterilization sea water adding 2%MES nutritional solution.
Preferably, in step S2, after the carpospore cyst on thallus is reached maturity, thallus is taken out at room temperature cloudy
Dry, then thallus is placed in sterilization sea water 1.5～2.5h, stimulate carpospore cyst to diffuse out carpospore；Every day is repeated once, directly
To collecting sufficient amount of carpospore.
Preferably, in step S3, when the length of carpospore formation germ tube and germ tube reaches 3-5 times of carpospore diameter
Time, the carpospore taking single sprouting carries out single culture.The length forming germ tube and germ tube carpospore reaches carpospore
Carry out single culture during 3-5 times of diameter, the probability that obtain heterozygosis carpospore can be greatly improved.
Preferably, in step S4, monoclonal filamentous algae falls and diffuses conchospore and comprise the steps: to take list after induction
The algae section that the filamentous algae of clone falls is enlarged cultivating, and takes a small amount of filamentous after 25～35d, and the algae section taking this filamentous is placed in
Temperature 25 ± 1 DEG C, photon flux density 20-30 μm ol m-2·s-1, quiescent culture under conditions of photoperiod 10L:14D, cultivate
Nutrition algal filament to more than 50% is reached maturity, and forms conchosporangia；Take out half and contain the algal filament of conchosporangia in temperature
Spend 19 ± 1 DEG C, photon flux density 40-50 μm ol m-2·s-1, cultivate under conditions of photoperiod 10L:14D, to ripe shell spore
Ascus branch release conchospore, in culture fluid, after the culture fluid (with 200 mesh sieve thin,tough silk) containing conchospore is filtered out impurity, both must
Conchospore.
Preferably, the process of described amplification culture falls for taking filamentous algae, with the list of sterilization on the plastic base plate of sterilization
Face blade chopping, the algae segment length after chopping is advisable with 1-2mm, and all algae sections are transferred to the triangle containing 100ml culture fluid
Amplification culture in flask, condition is temperature 19 ± 1 DEG C, photon flux density 40-50 μm ol m-2·s-1, photoperiod 10L:14D,
Quiescent culture.
Preferably, in step S5, select the chimeric thallus single culture containing four color lumps, when a certain color lump length extremely
0.8～1.2cm long time, cut single culture and induce it to diffuse monospore, as this color lump through induction still put without monospore
Dissipate, then this color lump sea snail enzymes liquid is processed and obtain isolated somatic cell.
Preferably, in step S5, described monospore or isolated somatic cell, when sprouting to 0.8～1.2cm, carry out single training
Support.
Preferably, described hermaphroditism Thallus Porphyrae is Porphyra yezoensis Ueda.
Compared with prior art, there is advantages that
1, the present invention solves the details such as the hybridization of hermaphroditic spot laver thallus and the qualification of heterozygosis filamentous
Problem, and construct the mapping population Double-haploid population of Porphyra yezoensis Ueda genetic map on this basis；
2, currently without the structure of open source information report hermaphroditism Thallus Porphyrae Porphyra yezoensis Ueda genetic linkage maps, and map
The structure of colony is the basis of genetic map construction, builds hermaphroditism Thallus Porphyrae Double-haploid population by the present invention, thus is
Build hermaphroditism Thallus Porphyrae (e.g., Porphyra yezoensis Ueda) genetic linkage maps to lay the foundation.
3, genetic linkage maps can instruct the extensive assembling of genome, also can position the QTLs of important economical trait simultaneously
Site, lays the foundation for clone's critical function gene, therefore the present invention is the basis of Porphyra yezoensis Ueda genomics research.
4, at present without the complete collection of illustrative plates of open source information report Porphyra yezoensis Ueda full-length genome, the achievement of the present invention will be for full genome
The extensive assembling of group provides powerful.
Accompanying drawing explanation
Fig. 1 is thallus (age in days 30d, the scale of Porphyra yezoensis Ueda wild chromaticity system PY-LS and red mutation strain PY-HT
5cm)；
Fig. 2 is that the filamentous algae sprouted after Porphyra yezoensis Ueda carpospore sinks to the bottom falls；
Fig. 3 is the chimeric thallus seedling that Porphyra yezoensis Ueda has the combination of parents' color；
Fig. 4 is the filamentous that Porphyra yezoensis Ueda is fitted together to four color lumps of thallus；
Fig. 5 is Porphyra yezoensis Ueda four color lump color-sectored blade.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.Following example will assist in those skilled in the art
It is further appreciated by the present invention, but limits the present invention the most in any form.It should be pointed out that, to those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make certain adjustments and improvements.These broadly fall into the guarantor of the present invention
Protect scope.
Embodiment
The selection of 1.1 Porphyra yezoensis Ueda hybrid strain thallus (gametocyte) and cultivation
Porphyra yezoensis Ueda as hybrid strain should have obvious morphology labelling, be typically chosen there are naked eyes can obvious district
Point the different frond of color, this programme is with Porphyra yezoensis Ueda wild chromaticity system PY-LS (Fig. 1, left figure) and red mutation strain PY-
Illustrate as a example by HT (Fig. 1, right figure).In addition, considering between parent while polymorphic differences and fertility, should try one's best
Choose sibship relatively far away from, genetic diversity is big and molecular marker polymorphism is strong material as hybrid strain, be beneficial to
Carry out location and the clone of merit gene.
Weigh the parent filarnentous body (sporinite) of 100mg weight in wet base, put on the plastic base plate of sterilization, with the single-edge blade of sterilization
Being chopped into short algae section, length, at about 1mm, is transferred in the glass culture dish of a diameter of 90mm cultivate, with the side crossed
Formula shakes up, and makes algae section be uniformly distributed in ware.Culture dish containing algae section is placed in temperature 25 ± 1 DEG C, photon flux density 20-
30μmol m-2·s-1, quiescent culture under conditions of photoperiod 10L:14D.Developmental state, about about 30d is checked every 5d, can
See that the nutrition algal filament of more than 50% is reached maturity, form conchosporangia.
From culture dish, take out the algal filament of half with tweezers, put in the gas filling bottle containing 200ml sterilization sea water and cultivate, separately
Put into outward one vinylon rope (long 3-4cm) to adhere to for conchospore, cultivation temperature 19 ± 1 DEG C, photon flux density 40-50 μm ol
m-2·s-1, photoperiod 10L:14D.Under the stimulation of lower temperature, ripe conchosporangia forms double points of spores further, releases
Being put in culture fluid i.e. conchospore, conchospore is attached on vinylon rope under the effect of current.Take out the dimension of attachment conchospore
Nylon rope moves to new inflation culture in glassware.Conchospore is sprouted on vinylon rope, about 30d duration to about 1cm little lobate
Body, takes continuation air-charging incubation from vinylon rope by it, chooses healthy complete and immature thallus and carry out list during to 40d
Cultivate, cultivate to thallus ending and initially form anthreid.Culture fluid used is the sterilization sea water rich in MES nutritive salt
(Wang Sujuan, Zhang Xiaoping, Xu Zhidong, etc. porphyra haitanensis trophocyte and the research of Protoplast cuhnre. Oceanologia et Limnologia Sinica,
1986.17 (3): 217-221.), every 5d changes the culture fluid of half.
The hybridization of 1.2 spot laver thallus and the collection of carpospore
Select each 1 of the parents' thallus (PY-LS and PY-HT) forming anthreid of health, put and sterilize containing 500ml
The gas filling bottle of sea water is mixed, makes the two hybridize, cultivation temperature 19 ± 1 DEG C, photon flux density 40-50 μm ol m-2·
s-1, photoperiod 10L:14D.The formation of the female sex cell carpogonium of spot laver thallus is later than the formation of anthreid, and
The two mixing is grown, and pretends the spot laver thallus for hybrid strain and can discharge sperm as male parent simultaneously, also can simultaneously
Accept sperm as female parent, i.e. self-fertilization and allogamy occurs simultaneously.Meanwhile, carpogonium is often in the edge shape of anthreid in blocks
Become, therefore the allogamy to be far above of autogamous probability.
During mixed culture, observed the formation of carpospore cyst on parents' thallus every 2 days, when being found to have a large amount of fruit
When Sporangium is formed, by parents' thallus separately, respectively by different gas filling bottle single culture, until the fruit on respective thallus
Sporangium is reached maturity.Thallus taking-up absorbent paper is blotted surface moisture, and at room temperature dry in the shade thallus about 30min, then
Thallus is placed in the culture dish containing 50ml sterilization sea water, stimulates carpospore cyst to diffuse out carpospore, by thallus after 2h
Put back in gas filling bottle and cultivate.Culture dish is put check whether there is carpospore under inverted microscope diffuse, put when being found to have carpospore
When dissipating, the carpospore diffused is made to be uniformly distributed in culture dish with suction pipe piping and druming, and quiescent culture.Every day is repeated once, until
Collect sufficient amount of carpospore.
1.3 Porphyra yezoensis Ueda carpospores and the cultivation of filamentous
In the incubation of carpospore, every day microscopy its sprout situation, when observing that carpospore forms germ tube and sprouting
Send out the length of pipe when reaching 3-5 times of carpospore diameter, with homemade capillary glass tube (caliber 30-50 μm) by single sprouting
Carpospore sucking-off and containing 15ml sterilization sea water teat glass (15 × 200mm) in single culture so that it is sprout into one
Monoclonal filamentous algae falls.The carpospore sprouted in order to ensure each sucking-off one, can blow to the liquid of sucking-off carry glass
On sheet, check under the microscope.Simultaneously in order to prevent cross-contamination, often inhale the capillary glass tube that once should more renew, it is ensured that every
It is monoclonal that the filamentous algae grown up in individual test tube falls.Standing training will be placed on test tube rack containing the test tube sprouting carpospore
Supporting, condition of culture is with 1.2, and period is changed without culture fluid.Cultivate about 30d and examine visible carpospore in dividing that sprouting is formed
Branch filamentous, continues cultivation to the algae that can form a diameter 3-5mm during 50d and falls.The growth that filamentous algae falls in test tube has
Three types, including adherent, sink to the bottom (Fig. 2) or swim on liquid level.
The separation of 1.4 Porphyra yezoensis Ueda heterozygosis filamentouss and qualification
The heterozygosis carpospore diffused after the carpospore diffused after self-fertilization and allogamy is equal in terms of form and size
Cannot with the naked eye distinguish, thus with capillary glass tube draw single carpospore when can only randomization.Two kinds of carpospores sprout
The filamentous become there will be difference in color.If the PY-LS that hybrid strain is wild color and red PY-HT, because wild
It is dominant for adding lustre to relative to redness, then the heterozygosis carpospore diffused from PY-HT thallus can sprout into the heterozygosis of wild color
Filamentous (color is with the filamentous of PY-LS), and carpospore of isozygotying can sprout into the filamentous that isozygotys (the same PY-HT of color of redness
Filamentous)；If the carpospore collected from PY-LS thallus, either still the isozygotying of heterozygosis, all sprout into wild
The filamentous algae of color falls.
In order to verify further, heterozygosis filamentous algae to be verified need to be fallen and take out, with tweezers gently by algae from test tube
Falling to tearing up, and transfer to amplification culture in the conical flask containing 50ml culture fluid, condition of culture is with 1.2, and about about 30d takes few
Amount filamentous, the method according to 1.1 obtains the conchospore of heterozygosis filamentous to be verified.Difference is, only puts a root length about
The vinylon rope of 1cm, its purpose is to detect whether to diffuse conchospore.When conchospore starts to diffuse, every morning 11:
00-11:30 divides the silk cover filtering of culture fluid 200 mesh, and conchospore can be by the micropore on bolting silk, and filamentous is then blocked
On bolting silk.The conchospore quiescent culture in the culture dish of diameter 90mm that will be collected by filtration, makes conchospore base portion be attached to
Ware bottom growth.Filamentous on bolting silk is then returned in gas filling bottle, continues to stimulate it to diffuse conchospore.
Conchospore is quiescent culture in culture dish, and condition of culture is with 1.2, and period changes weekly half culture fluid.Cultivate 30d
The thallus of conchospore germination seen from the naked eyes of left and right, length about 5mm.Choose thallus and be placed on microscope slide use microscopy,
Record chimeric type and quantity.If it find that the thallus of more than 90% is the chimeric thallus (figure containing parents' color
3), then illustrate that this filamentous is heterozygosis filamentous, can be used for the structure of Double-haploid population.
The structure of 1.5 Porphyra yezoensis Ueda Double-haploid population
Select the F containing 4 color lumps1Chimeric thallus, every chimeric thallus single gas filling bottle cultivation, bar
Part is with 1.2.When a certain color lump length on chimeric thallus is to when about 1cm is long, is cut single culture, put into one simultaneously
The vinylon rope of 3cm length adheres to for monospore.If this color lump does not diffuse monospore, then its thallus sea snail enzymes liquid is processed
Obtain isolated somatic cell (Yan Xinghong, Liu Xinyi, kind thunderclap. the growth of spot laver thallus cell and differentiation. aquatic product journal,
2004(02):145-154.).By above-mentioned monospore or isolated somatic cell when it is sprouted to about 1cm, the most single cultivation,
The filamentous that isozygotys (strain) of each color lump on every chimeric thallus is obtained eventually through self-fertilization, method same 1.3, thus
It is built into Double-haploid population (Fig. 4).
4 color lumps are selected to be fitted together to thallus as the reason building Double-haploid population: the filamentous of Porphyra yezoensis Ueda is two times
Sporinite, the conchospore diffused also is diploid, and initial twice division of conchospore is meiosis, forms four subtrahends and divides
Splitting the unseparated four molecule bodies of daughter cell, spot laver thallus is then by being fitted together to that four haploid cells continuation divisions are formed
Thallus, but in actual growth course, four cells not necessarily normal development can form the chimera of 4 color lumps,
Therefore composition F1 is fitted together to thallophytic color lump quantity from 1-4.Only select the chimera of 4 color lumps, meiosis could be produced
Raw different genotype is integrally incorporated in Double-haploid population, selects to cause some can not less than the chimera of 4 color lumps
The disappearance of the genotype of normal development, thus destroy the hereditary constitution of Double-haploid population, it is possible to cause serious inclined separation
Phenomenon, affects the accuracy of genetic mapping.In the building process of porphyra haitanensis genetic linkage maps, the molecular marker partially separated is high
Reaching 29.3%, its reason is probably and does not select 4 color lump chimeras (to be fitted together to thallus from 50 F1 as the material building colony
On be separated to 166 color lumps) (Xie, C., C.Chen, Y.Xu wait .Construction of a genetic linkage
map for Porphyra haitanensis(Bangiales,Rhodophyta)based on sequence-related
amplified polymorphism and simple sequence repeat markers.Journal of
Phycology, 2010.46 (4): 780-787.), average each chimera has 3.3 color lumps.
The nomenclature principle of each strain of Double-haploid population: chimera numbering+color lump numbering+color lump color, chimera is numbered
For 1-50 or more than, represent containing the chimeric numbering of F1 of 4 color lumps；Color lump is numbered according to 4 color lumps on chimeric thallus
Linear arrangement order from base portion to ending, numbering 1-4, color lump 1 is positioned at chimeric thallophytic base portion, and color lump 4 is positioned at chimeric leaf
The ending of shape body；Color lump color letter representation, W is the wild color as PY-LS, and R is the redness as PY-HT, W '
With R ' is respectively the restructuring color formed after recombinating.If the numbered 36-3R of certain strain, then it represents that this strain is from selecting
36th 4 color lumps are fitted together to thallus, and this color lump position on chimera is positioned at the 3rd piece of number from the bottom up, and R represents this color lump
Color be red (Fig. 5).
Claims (10)
1. the method that a hermaphroditism Thallus Porphyrae Double-haploid population is set up, it is characterised in that described method comprises the steps:
S1, choose thallus color distortion obvious hermaphroditism thallus of porphyra as hybrid strain；
S2, choose formed anthreid parents' thallus mixed culture, to parents' thallus, begin with the shape of carpospore cyst
Cheng Shi, separates single culture by parents' thallus, until the carpospore cyst on respective thallus is reached maturity, collects carpogonium respectively
Son；
S3, when carpospore formed germ tube time, take the carpospore single culture of single sprouting so that it is sprout into one monoclonal
Filamentous algae falls；
S4, cultivate described monoclonal filamentous algae and fall the conchospore that diffuses after induction, to macroscopic F1Thallus, aobvious
Micro-Microscopic observation F1Thallophytic color and color lump quantity, such as the F of more than 90%1Thallus is the chimeric leaf containing parents' color
Shape body and color lump number are 2 pieces, 3 pieces or 4 pieces, then this monoclonal filamentous algae falls is thread by the heterozygosis of biparent cross
Body, its F1The chimeric thallus having four color lumps in Dai can be used for building Double-haploid population；
S5, from the chimeric thallus containing four color lumps, separate single color lump, take the monospore that this color lump diffuses after induction
Or isolated somatic cell, cultivate to macroscopic thallus, take single cultivation, obtain eventually through this single thallus self-fertilization
Obtain every four color lumps and be fitted together to the pure lines filamentous of each color lump on thallus, thus set up into Double-haploid population.
2. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S2
In, the parents' thallus having formed anthreid obtains as follows: the algae section taking parent filarnentous body is placed in temperature 25
± 1 DEG C, photon flux density 20-30 μm ol m-2·s-1, quiescent culture under conditions of photoperiod 10L:14D, culture fluid is for adding
Adding the sterilization sea water of 2%MES nutritional solution, the nutrition algal filament of cultivation to more than 50% is reached maturity, and forms conchosporangia；Take out
Half contains the algal filament of conchosporangia in temperature 19 ± 1 DEG C, photon flux density 40-50 μm olm-2·s-1, photoperiod 10L:
Cultivating under conditions of 14D, culture fluid is the sterilization sea water adding 2%MES nutritional solution, to ripe conchosporangia release conchospore
In culture fluid；The leaflet that culture fluid containing conchospore continues to cultivate to this conchospore germination is grown up, and to lobate
Body ending initially forms anthreid,.
3. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S2
In, the cultivation temperature of described mixed culture 19 ± 1 DEG C, photon flux density 40-50 μm ol m-2·s-1, photoperiod 10L:14D,
Culture fluid is the sterilization sea water adding 2%MES nutritional solution.
4. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S2
In, after the carpospore cyst on thallus is reached maturity, thallus is taken out and at room temperature dries in the shade, then thallus is placed in sterilization sea
In water 1.5～2.5h, carpospore cyst is stimulated to diffuse out carpospore；Every day is repeated once, until collecting sufficient amount of carpogonium
Son.
5. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S3
In, when the length of carpospore formation germ tube and germ tube reaches 3-5 times of carpospore diameter, take the carpospore of single sprouting
Single culture.
6. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S4
In, monoclonal filamentous algae falls and diffuses conchospore and comprise the steps: to take the algae that monoclonal filamentous algae falls after induction
Section is enlarged cultivating, and takes a small amount of filamentous after 25～35d, and the algae section taking this filamentous is placed in temperature 25 ± 1 DEG C, photon flux
Density 20-30 μm ol m-2·s-1, quiescent culture under conditions of photoperiod 10L:14D, cultivate the nutrition algal filament to more than 50%
Reach maturity, form conchosporangia；Taking out half and contain the algal filament of conchosporangia temperature 19 ± 1 DEG C, photon flux is close
Degree 40-50 μm ol m-2·s-1, cultivate under conditions of photoperiod 10L:14D, to ripe conchosporangia release conchospore to training
In nutrient solution, after filtering and impurity removing, both obtained conchospore.
7. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 6 is set up, it is characterised in that described expansion
The process cultivated falls for taking filamentous algae, by the single-edge blade chopping of sterilization, the algae section after chopping on the plastic base plate of sterilization
Length is advisable with 1-2mm, and all algae sections are transferred to amplification culture in the conical flask containing 100ml culture fluid, and condition is temperature
Spend 19 ± 1 DEG C, photon flux density 40-50 μm ol m-2·s-1, photoperiod 10L:14D, quiescent culture.
8. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S5
In, select the chimeric thallus single culture containing four color lumps, when a certain color lump length to 0.8～1.2cm is long, cut
Single culture also induces it to diffuse monospore, such as this color lump through induction still without temperature, then by this color lump sea snail enzymes liquid
Process and obtain isolated somatic cell.
9. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that step S5
In, described monospore or isolated somatic cell, when sprouting to 0.8～1.2cm, carry out single cultivation.
10. the method that hermaphroditism Thallus Porphyrae Double-haploid population as claimed in claim 1 is set up, it is characterised in that described female
Male consubstantiality Thallus Porphyrae is Porphyra yezoensis Ueda.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106857237A (en) * | 2017-03-01 | 2017-06-20 | 集美大学 | A kind of method of porphyra haitanensis Doubled haploid breeding |
| CN112690208A (en) * | 2020-12-18 | 2021-04-23 | 中国科学院海洋研究所 | Method for constructing Double Haploid (DH) sporophyte population in undaria pinnatifida |
| CN112913678A (en) * | 2020-05-15 | 2021-06-08 | 中国科学院海洋研究所 | A method for preparing permanent F from Undaria Pinnatifida2Methods of sporozoite population |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029160A1 (en) * | 1997-12-12 | 1999-06-17 | Northeastern University | STRAIN MANIPULATION AND IMPROVEMENT IN THE EDIBLE SEAWEED $i(PORPHYRA) |
| CN101558738A (en) * | 2009-04-17 | 2009-10-21 | 中国海洋大学 | Porphyra haitanensis molecular marker-assisted selection breeding method |
| KR20120070805A (en) * | 2010-12-22 | 2012-07-02 | 한국원자력연구원 | A mutant porphyra lucasii 500g having higher resistance against oxidizing stress or higher antioxidant activity, and preparation method thereof |
-
2016
- 2016-06-16 CN CN201610429722.8A patent/CN106069717B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029160A1 (en) * | 1997-12-12 | 1999-06-17 | Northeastern University | STRAIN MANIPULATION AND IMPROVEMENT IN THE EDIBLE SEAWEED $i(PORPHYRA) |
| CN101558738A (en) * | 2009-04-17 | 2009-10-21 | 中国海洋大学 | Porphyra haitanensis molecular marker-assisted selection breeding method |
| KR20120070805A (en) * | 2010-12-22 | 2012-07-02 | 한국원자력연구원 | A mutant porphyra lucasii 500g having higher resistance against oxidizing stress or higher antioxidant activity, and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 刘美君等: ""条斑紫菜种内杂交优良品系的筛选与特性分析"", 《中国水产科学》 * |
| 吴宏肖等: ""坛紫菜与Pyropia radi种间杂交重组优良品系的选育与特性分析"", 《水产学报》 * |
| 张源等: ""自然条件下的坛紫菜四分体发育与性别表型观察"", 《水产学报》 * |
| 王凤霞等: ""褐绿色型与野生色型坛紫菜杂交产生嵌合体及其选育"", 《集美大学学报(自然科学版)》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106857237A (en) * | 2017-03-01 | 2017-06-20 | 集美大学 | A kind of method of porphyra haitanensis Doubled haploid breeding |
| CN112913678A (en) * | 2020-05-15 | 2021-06-08 | 中国科学院海洋研究所 | A method for preparing permanent F from Undaria Pinnatifida2Methods of sporozoite population |
| CN112690208A (en) * | 2020-12-18 | 2021-04-23 | 中国科学院海洋研究所 | Method for constructing Double Haploid (DH) sporophyte population in undaria pinnatifida |
| CN112690208B (en) * | 2020-12-18 | 2022-06-07 | 中国科学院海洋研究所 | Method for constructing Double Haploid (DH) sporophyte population in undaria pinnatifida |
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