CN114208735A - Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology - Google Patents
Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology Download PDFInfo
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Abstract
The invention discloses a method for breeding a new rapid-growth strain of hong Kong oyster triploid by using a backcross breeding technology. Through the technical links of breeding the rapid growth line of the crassostrea hongkongensis and the diploid of the crassostrea, constructing the triploid induction line of the crassostrea hongkongensis, constructing the rapid growth line of the diploid of the crassostrea hongkongensis, culturing the rapid growth line of the diploid of the crassostrea yugensis, constructing the rapid growth line of the diploid of the crassostressostressostressostressostressostressostresson, and culturing the rapid growth line of the backcross triploid of the crassostresson and the diploid of the near-fragrant hybridized to carry out backcross and hybridization among different ploids, a new line of the backcross triploid rapid growth of the crassostresson hongkongensis obtained, the new line has the advantages of the triploid such as quick growth, obvious improvement of the survival rate of high salt area, stable 100 percent triploid rate and the like, has the advantages of pure crassons such as white meat, tough meat, thick shell and the like, and is an excellent new line suitable for commercial culture in the south China.
Description
Technical Field
The invention belongs to the technical field of shellfish genetic breeding in marine agriculture, and particularly relates to a method for cultivating a new rapid-growth strain of hong Kong oyster triploid by utilizing a backcross breeding technology.
Background
The hong Kong oyster is commonly called white oyster, is an important oyster cultivation variety for coastal cultivation in Guangdong, Guangxi and Hainan in China, has the advantages of delicious taste, elastic and tough meat quality and the like, and is widely popular with people in south China. The annual output of the oyster in 2020 is about 179 tons, which accounts for 32.97% of the national oyster output, and compared with the annual output of 2019, the annual output has a descending trend, mainly due to reasons of channel sea area cleaning, long culture period, reduction of suitable culture sea area and the like, the price of the oyster is gradually increased, the market price is increased to 10 yuan per kilogram (more than two specifications of 4), and the oyster is a high-end variety of the oyster in China. Since the hong Kong oyster triploid starts to be popularized by the team in 2018, although the growth speed of the hong Kong oyster triploid fed back by farmers is improved relative to that of the diploid, the hong Kong oyster triploid still has a slow growth speed, still needs at least 2 years to reach the marketing specification, the cultivation risk is high, and the death rate in a high-salt time period is high. The farmer needs to further improve the growth and high-salt resistance of the triploid of the crassostrea hongkongensis, so that the introduction of a gene with fast growth and high-salt resistance is needed. The ostrea rivularis is an oyster species with wider salt tolerance range and stronger disease resistance and stress resistance, but has yellow meat quality and poorer taste and sharply reduced cultivation range. Through a backcross breeding technology, accumulated hybrid advantages and backcross advantages can be generated, and the high-salt resistance of the crassostrea hongkongensis is improved in an accumulated mode.
The new species with excellent characters are cultured by a backcross breeding technology and are common in aquatic animals, for example, the first filial generation of the crassostrea hongkongensis and the crassostrea gigas is backcrossed to produce seeds, and a high-salt-resistant 'No. 1' new species in south China is obtained; the hybrid offspring of the bay scallop and the purple scallop and the bay scallop are backcrossed to produce the seed, and the new variety of the Qingnong No. 2 with fast growth, low mortality and black shell is obtained. However, the backcross breeding may cause gene pollution to natural populations, influence the diversity of marine organisms, and cause disadvantages and disadvantages which are not convenient to measure. The triploid has triploid advantages compared with diploid, and has the advantages of poor fertility, fast growth, high survival rate and the like, and the triploid fertile individuals have extremely low survival rate of self-reproduction filial generation and basically have no influence on the diversity of marine organisms.
Disclosure of Invention
The invention aims to provide a method for cultivating a new triploid rapid-growth strain of crassostrea hongkongensis, which has the advantages of rapid growth, extremely poor fertility, high survival rate, good quality and 100 percent of triploid rate, by utilizing a backcross technology.
The method comprises the steps of breeding a rapid growth line of the diploid of the Ostrea rivularis and a rapid growth line of the diploid of the Ostrea hongkongensis, continuously constructing a triploid line of the Ostrea rivularis, a tetraploid line of the Ostrea rivularis and a fragrant male hybrid diploid, and finally carrying out artificial fertilization by using the tetraploid male of the Ostrea rivularis and a fragrant male hybrid first generation female, so as to obtain a new triploid backcross rapid growth line of the Ostrea hongkongensis. The invention accumulates the advantages of backcross and triploid, cultivates and obtains a new triploid strain with fast growth, poor fertility, high salt tolerance and high survival rate, provides a solution for responding to the requirement of the hong Kong oyster industry, and improves the yield and output value, strengthens the industry, improves the income of fishermen, reduces the cultivation risk and the like, and is the innovation point of the invention.
The invention discloses a method for cultivating a new rapid-growth strain of hong Kong oyster triploid by using a backcross technology, which is characterized by comprising the following steps of:
A. respectively breeding a hong Kong oyster diploid rapid growth line and a near river oyster diploid rapid growth line;
B. b, taking the near river oyster diploid fast growth line of the step A as a parent to obtain a near river oyster triploid induction population line;
C. using the diploid rapid growth line of the ostrea rivularis as a male parent, and using fertile individuals in the triploid induction group line of the ostrea rivularis as female parents to obtain a tetraploid rapid growth line of the ostrea rivularis through induction;
D. using the quick growth line of the near river oyster diploid as a male parent and using the quick growth line of the hong Kong oyster diploid as a female parent, and performing interspecific hybridization to obtain a hybrid oyster diploid quick growth line;
E. the tetraploid fast growing line of the crassostrea rivularis is taken as a male parent, and the diploid fast growing line of the hybridized crassostrea rivularis is taken as a female parent, and the hybridization is carried out to obtain the triploid fast growing new strain of the hong Kong oyster.
Preferably, the method for breeding the hong Kong oyster triploid fast-growing new strain by utilizing the backcross technology comprises the following steps:
A. respectively collecting 3-5 different geographical group lines of the diploid of the crassostrea hongkongensis and the diploid of the crassostrea crassoensis, continuously breeding for more than 4 generations by taking growth and survival as target characters, and respectively breeding to obtain the rapid growth line of the diploid of the crassostrea hongkongensis and the rapid growth line of the diploid of the crassostrea crassoensis;
B. respectively taking the quick growth line of the ostrea rivularis diploid in the step A as a parent, and after 2N male parent and 2N female parent fertilization, using CB to inhibit pb2 release to obtain a ostrea rivularis triploid induction line;
C. taking the near river oyster diploid in the step A as a male parent, taking a fertile individual in the near river oyster triploid induction population in the step B as a female parent, and inhibiting the release of the polar body after fertilization by 2N male parent multiplied by 3N female parent to obtain a near river oyster tetraploid rapid growth line through induction;
D. taking the quick growth line of the near river oyster diploid in the step C as a male parent, taking the quick growth line of the hong Kong oyster diploid as a female parent, and obtaining a hybrid oyster diploid quick growth line after interspecific hybridization of 2N male parent and 2N female parent fertilization;
E. and (4) hybridizing the tetraploid rapid growth line of the ostrea rivularis in the step (C) with the diploid rapid growth line of the hybridized oysters in the step (D) as a female parent to obtain the triploid rapid growth new strain of the hong Kong oysters.
Further preferably, the method for cultivating the rapid-growth new strain of the hong Kong oyster triploid by utilizing the backcross technology comprises the following specific steps:
A. breeding the diploid rapid growth line of the hong Kong oyster and the ostrea rivularis: collecting 3-5 different wild populations of Crassostrea hongkongensis and Crassostrea rivularis respectively, analyzing the interaction between genotype and environment type by taking growth and survival as targets, and screening out the populations with the best performance respectively; self-breeding the optimal population, continuously breeding 4 generations according to the seed reserving rate of 10 percent for each generation by taking growth and survival as breeding targets, and respectively breeding a Hongkong oyster diploid rapid growth line and a crassostrea gigas diploid rapid growth line;
B. constructing a crassostrea rivularis triploid induction line: taking individuals with good shell type and large full weight of the ostrea rivularis diploid rapid growth line in the A, arranging the individuals from large to small according to shell height, taking the first 10% of the individuals as parents, dissecting and identifying males and females after temporary culture, artificially fertilizing, inducing and treating by cytochalasin B when about 50% of a first polar body Pb1 is released after fertilization, and incubating and selecting excellent larvae, namely the ostrea rivularis triploid induction line;
C. constructing a tetraploid rapid growth line of the ostrea rivularis: and (4) respectively taking individuals with good shell type and large full weight of the ostrea rivularis diploid rapid growth line in the step a and the ostrea rivularis triploid induction line in the step b, and identifying the triploid ploidy after temporary culture. Respectively screening individuals with 10% shell height as parents; taking a diploid as a male parent and a triploid fertile female individual as a female parent, carrying out artificial fertilization, when about 50% of first polar body is released, inducing by using 6-dimethylaminopurine (6-DMAP), putting into seawater for hatching, selecting D-type larvae and detecting the tetraploid rate, and culturing and cultivating in an excellent sea area, wherein the first polar body is an ostrea rivularis tetraploid induction line; screening individuals with larger overall weight and uniform size from the tetraploid induction line of the ostrea rivularis, identifying the tetraploid by a flow cytometer, arranging the tetraploid according to the shell height from large to small, taking the first 10 percent as a parent, carrying out artificial fertilization, selecting excellent larvae and identifying the tetraploid rate, thus obtaining the rapid growth line of the ostrea rivularis tetraploid;
D. constructing a rapid growth line of the diploid of the hybridized oyster: respectively taking individuals with large full weight and good shell shape of the near river oyster diploid fast growing line and the hong Kong oyster diploid fast growing line in the step a; after temporary rearing, arranging the shell heights from large to small, selecting the first 10% of individuals as parents, dissecting and identifying males and females, taking ostrea rivularis as a male parent and hong Kong oysters as a female parent, performing artificial fertilization, and breeding larvae, thus obtaining the hybrid oyster diploid rapid growth line;
E. cultivating a backcross triploid rapid growth line: and (4) respectively taking individuals with heavier weight and better shell shape of the tetraploid rapid growth line of the ostrea rivularis in the step (c) and the diploid rapid growth line of the hybrid oyster in the step (d), identifying the tetraploid after temporary rearing, carrying out artificial fertilization by using a tetraploid male and the diploid female of the hybrid oyster, hatching, selecting and identifying the triploid rate, and finely cultivating larvae and juvenile mollusks, which are the new strains for rapid growth of the triploid of the hong Kong oyster.
Through the links, the novel triploid rapid-growth strain of the hong Kong oyster is obtained, has the advantages of faster growth and higher salt tolerance, and has the advantages of poor fertility, high survival rate, white and elastic meat, delicious taste, capability of being listed all year round, 100 percent triploid rate and the like of the triploid and the hong Kong oyster.
Preferably, the different wild populations of crassostrea hongkongensis and crassostrea rivularis in the A are populations which are collected in the field, have different geographic regions and high variation degree. Therefore, the method can ensure that the genetic variation among different groups is large, and can breed the group with excellent real performance, thereby laying the foundation for the follow-up work.
Preferably, the crassostrea rivularis triploid induction line in the step B is constructed, the conditions of ovum soaking, fertilization and larva cultivation seawater are 29-31 ℃ and 15ppt of salinity, a pre-experiment is required before induction, the release rule of an polar body is discovered, and the high triploid rate is conveniently induced. The induction concentration of CB is 0.50mg/L, the induction starting time is determined according to the polar body release rule, and after fertilization, when about 50% of Pb1 in the first polar body is released, Cytochalasin B (CB) is used for induction treatment for 30 min.
Preferably, the rapid tetraploid growth line of the ostrea rivularis in the step C is constructed, the conditions of ovum soaking, fertilization and larva cultivation seawater are 29-31 ℃ and 15ppt of salinity, a pre-experiment is required before induction, and the tetraploid rate is high. The triploid ovum needs to be strictly screened, so that the maturity of the ovum is high, the substance in the ovum nucleus is rich, the size is uniform, and the ovum is free from nicking. The induction onset time is adjusted according to the release of polar bodies, and both advancing and retarding can result in high teratogenesis and low larva survival rate. When the first polar body released about 50%, it was induced with 100mg/L of 6-DMAP (6-dimethylaminopurine) for 65 min. The tetraploid induction line of the ostrea rivularis is selected for only one generation subsequently, the genetic diversity of the tetraploid is reduced and the survival rate of filial generation is low due to multiple selection generations.
Preferably, the rapid growth line of the hybrid oyster diploid in the step D is constructed, and male and female individuals, a fresh water cleaning container and the like are strictly separated in the artificial fertilization process, so that accidental fertilization is avoided. Larvae need to be identified as true ostrea rivularis sperm and crassostrea hongkongensis ovum hybrids using COI and ITS-2 nuclear genes. The offspring after fertilization of the ostrea rivularis sperm and the crassostrea hongkongensis ovum has the advantages of faster growth and higher survival rate than the parents, and the meat quality and the taste are similar to those of the female parent crassostrea hongkongensis, so the hybrid fast growth line is called.
Preferably, the hong Kong oyster triploid is bred by a new rapid-growth strain in E, tetraploid sperms and diploid ova are hybridized to generate triploids, the ostrea rivularis male parent and the heterozygote female parent are backcross breeding, and the advantages of the triploids and the backcross advantages are combined. In the artificial fertilization process, the operation is strictly carried out according to the flow, so as to avoid accidental fertilization. Tetraploids require flow cytometry to identify the ploidy of the adductor muscle and sperm.
Preferably, the A, B, C, D and E larva fine breeding technology comprises the steps of adjusting bait types at different development stages (D type larva stage bait is golden algae, shell top larva stage bait is golden algae and cloud microalgae mixed bait, and adhered hornbeam, Platymonas subcordiformis and cloud microalgae mixed bait), adjusting breeding density according to growth, monitoring temperature and salinity, changing water by 20% every 3 days, and the like, so that larva breeding success rate is ensured, and death rate is reduced.
Based on the difficult problem of industrial neckerchief that the growth cycle of the triploid of the hong Kong oyster is still relatively long and the high-salt resistance is poor, the invention obtains the tetraploid rapid growth line of the Ostrea rivularis and the diploid rapid growth line of the hybrid oyster by improving the parents, and combines the advantages of backcross and triploid of the oysters to cultivate the triploid rapid growth new strain of the hong Kong oyster. The invention overcomes the defect that the triploid fertility of the hong Kong oyster cannot be directly improved, and finally indirectly obtains the triploid rapid growth line of the hong Kong oyster by improving the rapid growth of the tetraploid male parent and the diploid female parent. The crassostrea hongkongensis triploid obtained by the invention can quickly grow a new strain, the growth speed is obviously improved, the high salt resistance is better, the disease resistance and stress resistance are stronger, and the traditional triploid advantages of stable 100 percent triploid rate, delicious meat, extremely poor fertility, high survival rate, capability of coming into the market all the year round and the like are also achieved. The obtained rapid growth line of the hong Kong oyster triploid is 22.48 to 26.37 percent higher than the growth of pure hong Kong oyster triploid (which is not improved) in low-salt sea areas, and the survival rate is 0.41 to 9.63 percent; the growth in high-salt sea areas is improved by 11.13-28.23%, and the survival rate is improved by 6.09-35.56%. The invention also provides a new idea for solving the difficult problems of the hong Kong oyster industry and protecting the property rights of excellent new strains, and provides guarantee and support for the continuous healthy development of the industry and the joyful aquatic breeding industry.
The method has the advantages of strong operability, relatively easy strain property right protection, large popularization potential and the like.
Description of the drawings:
FIG. 1 is a technical roadmap for the present invention;
the specific implementation mode is as follows:
the following examples are provided to illustrate the method for breeding a new rapidly growing line of the triploid crassostrea hongkongensis by using the backcross breeding technology, but the invention is not limited thereto.
Example 1
a, breeding diploid rapid growth lines of Crassostrea hongkongensis and Crassostrea rivularis: collecting 200 individuals of Guangdong Shenzhen population, Guangdong Yangjiang population, Guangdong Taishan population, Guangxi North sea population, Guangxi defense town harbor population, Guangdong Ostring Shenzhen population, Guangdong Taishan population and Guangxi Qinzhou population of the Ostrea in the wild, transporting to the southeast test field of the Zhanjiang ocean economic animal test station of the Chinese academy of sciences, and temporarily culturing for 7 days at the temperature of 29-31 ℃ and the salinity of 15ppt of a circulating seawater system; measuring the shell height, arranging the shell heights from big to small, taking individuals 10% of the shell height as parents, selfing in a group line to obtain offspring of each wild group, respectively arranging the offspring in 3 sea areas of Guangdong Zhuhai, Guangdong Taishan and Guangxi North sea after attachment, tracking and measuring the shell height, the total weight and the survival rate, carrying out genotype and environment type interaction analysis, and comprehensively comparing to obtain the group line with the best performance, namely the good colony of the Crassostrea hongkongensis and the good colony of the Ostrea crassoides; thirdly, in 5 months of 2014, 300 individuals of the good population of the hong Kong oyster and 300 individuals of the good population of the ostrea rivularis are respectively selected, temporarily cultured in a circulating seawater system for 7 days, and the individuals with the height of 10 percent of the shell are selected as parents to be respectively self-bred to obtain a next generation breeding line; and continuously breeding 3 generations (8 months per generation of sea), and respectively breeding in 2017 to obtain the Hongkong oyster diploid rapid growth line and the near river oyster diploid rapid growth line according to the seed reserving rate of 10% and the shell height as the breeding target.
b, constructing a ostrea rivularis triploid induction line: firstly, in 1 month of 2018, respectively taking 300 individuals with larger total weight and better shell shape from a near river oyster diploid rapid growth line, temporarily culturing for one week in a circulating water system (at the temperature of 29-31 ℃ and the salinity of 15ppt), then measuring the shell heights of the individuals, arranging the shell heights from large to small, and taking the first 10 percent of the individuals as parents; dissecting and identifying male and female, extruding egg, washing egg and soaking hydrated egg in sea water at 29-31 deg.c and salinity of 15 ppt. After more than 60% of the ovum is broken by the raw foam, extruding the sperms, screening the sperms with better activity under a microscope, and activating the sperms in the seawater for 10 min. Artificial fertilization, setting a pre-experimental group, and observing the release condition of the polar body under a microscope. Adding a proper amount of sperms into the ovum, and treating the ovum for 30min by using 0.50mg/L CB when about 50% of the first polar body Pb1 is released according to the polar body release rule of a pre-experimental group. After the treatment is finished, washing off the drugs, putting the washed drugs into seawater for hatching, screening D-shaped larvae, detecting the triploid rate of the larvae by using a flow cytometer, and keeping an induction group with the triploid rate of more than 90%. Finely cultivating larvae, adjusting the types, density and the like of baits according to the development of the larvae, and putting the larvae into an excellent sea area of the great Fengjiang of the North sea of Guangxi to cultivate and cultivate the larvae, namely the ostrea rivularis triploid induction line.
c, constructing a tetraploid rapid growth line of the ostrea rivularis: respectively taking 100 individuals and 300 individuals of the near-river oyster diploid rapid growth line in a and the near-river oyster triploid induction line in b in 2018 in 9 months, and temporarily culturing for one week in a circulating seawater system (with the temperature of 29-31 ℃ and the salinity of 15 ppt); identifying ploidy of all individuals in the triploid induction group, screening out triploid, identifying fertile triploid females, and arranging according to shell height from large to small. And simultaneously measuring the height of the male individual of the diploid fast growth line, wherein the height of the male individual is arranged according to the size. Taking the first 10% of individuals as parents, soaking triploid ova in seawater with the salinity of 15ppt at the temperature of 29-31 ℃ until the foaming is broken, and screening the sperms of diploid ostrea rivularis with the best activity. Artificial fertilization is carried out by diploid sperms and triploid ova, and a pre-experimental group is firstly arranged to know the polar body release rule. Thirdly, according to the release rule of the polar body in the preliminary experiment, when the first polar body releases about 50 percent, fertilized eggs are treated by 100 mg/L6-DMAP for 65min, after incubation, the fertilized eggs are preferably hatched, the tetraploid rate is detected, groups with the tetraploid rate exceeding 90 percent and the aberration rate lower than 10 percent are reserved, larvae are cultivated by a refined culture system, and excellent sea areas of the great Fengjiang in the North China sea in Guangxi are cultivated and cultivated on the sea, so that the induced line is the tetraploid induction line of the ostrea rivularis; and fourthly, after 6 months of marine culture (2 months in 2019), 300 individuals with large full weight and good shell shells are screened from a tetraploid induction line, the tetraploid is promoted to mature according to the result of the temperature accumulating effect of the ostrea rivularis, the tetraploid is identified after the gonad is mature, the tetraploid is arranged from large to small according to the shell height, the first 10 percent of the tetraploid is taken as a parent, artificial fertilization, incubation, optimization and identification of the tetraploid rate are carried out, tetraploid larvae are finely cultured, and the tetraploid is cultured and cultured in the sea, so that the tetraploid rapid growth line of the ostrea rivularis is obtained.
d, constructing a diploid rapid growth line of the hybridized oyster: respectively taking 200 individuals and 300 individuals with mature glands, large weight and good shell shapes from a near river oyster diploid rapid growth line and a hong Kong oyster diploid rapid growth line in a in 2018 in 4 months, temporarily culturing in circulating seawater (the temperature is 29-31 ℃ and the salinity is 15ppt) for one week, and reducing oxygen and running water to avoid self-discharge; measuring the shell height, arranging according to the size of the shell height, taking the first 10 percent of individuals as parents, identifying male and female, fertilizing by using ostrea rivularis sperms and crassostrea hongkongensis ova, strictly operating, and preventing accidental intraspecific fertilization. After artificial fertilization, hatching and optimizing in seawater, identifying the larvae as real hybridization by ITS-2, and culturing in the good sea area of the great Fengjiang river and the bamboo forest salt field in the North sea of Guangxi after the larvae are attached, thereby obtaining the rapid growth line of the hybridized oyster diploid.
e, culturing a triploid rapid growth line: in 8 months in 2019, respectively taking 200 individuals and 300 individuals with mature glands, heavier shell heights and better shell shapes of the hybrid oyster diploid rapid growth line from the near-river oyster tetraploid rapid growth line and the near-river oyster tetraploid rapid growth line in c, and temporarily culturing for 7 days in a circulating water system (at the temperature of 29-31 ℃ and the salinity of 15 ppt); secondly, after temporary rearing, dissecting and identifying tetraploid, using tetraploid as male parent and diploid as female parent to make artificial fertilization, hatching in seawater, selecting optimum and finely cultivating larva, so that the hong Kong oyster triploid fast growth line is obtained.
a. And b, c, D and e, namely adjusting the bait types in different development stages (the D-type larva stage bait is golden algae, the shell top larva stage bait is golden algae and cloud microalgae mixed bait, and the adhered mixture of chaetoceros, tetraselmis and cloud microalgae is added with water, adjusting the culture density according to the growth, monitoring the temperature and salinity, changing water by 20 percent every 3 days, and the like, so that the larva culture success rate is ensured, and the death rate is reduced.
f. Male tetraploid of Crassostrea hongkongensis (from the northern sea of Guangxi) and female diploid of Crassostrea hongkongensis (from the northern sea of Guangxi, without selective breeding) are hybridized to obtain triploid line of Crassostrea hongkongensis, which is used as a control group. And e, attaching larvae of the unmodified line of the hong Kong oysters in the control group and the triploid rapid growth line of the hong Kong oysters in the step e, respectively arranging the larvae in 2 sea areas of the great Fengjiang (low salt point, salinity variation range of 15-25ppt) and the bamboo forest (high salt point, salinity variation range of 25-33ppt) in the north China sea, and tracking and measuring the heights of shells and the survival rates of the larvae.
The results are shown in tables 1 and 2:
TABLE 1 Shell height comparison of the rapid growth line of the triploid hong Kong oyster and the unmodified line of the triploid hong Kong oyster in 2 different breeding sites
The letters on the same row are different and represent significant differences; the same row letters are identical and represent no significant difference.
TABLE 2 comparison of survival rates of triploid rapid growth lines of hong Kong oysters and pure variety triploid unmodified lines of hong Kong oysters at 2 different breeding sites
The letters on the same row are different and represent significant differences; the same row letters are identical and represent no significant difference.
The new triploid rapid-growth strain of the crassostrea hongkongensis is obtained by the cultivation of the steps and is arranged in 2 excellent culture sea areas of bamboo forest in the north sea of Guangxi and the great Fengjiang river in the north sea of Guangxi. The growth of 360 days is tracked and measured, and in the low-salt sea area-the point of the great Fengjiang in the North sea, the rapid growth line of the triploid of the hong Kong oyster is found to be 22.48 to 26.37 percent faster than the growth of the unmodified line of the triploid of the pure hong Kong oyster, the survival rate is 0.41 to 9.63 percent, and the growth is obviously improved. In the high-salt sea area, bamboo forest points, the rapid growth line of the hong Kong oyster triploid is 11.13 to 28.23 percent faster than that of the pure-breed hong Kong oyster triploid, the survival rate is improved by 6.09 to 35.56 percent, and the growth and high-salt resistance are obviously improved.
Claims (7)
1. A method for cultivating a new rapid-growth strain of hong Kong oyster triploid by using a backcross technology is characterized by comprising the following steps:
A. respectively breeding a hong Kong oyster diploid rapid growth line and a near river oyster diploid rapid growth line;
B. b, taking the near river oyster diploid fast growth line of the step A as a parent to obtain a near river oyster triploid induction population line;
C. using the diploid rapid growth line of the ostrea rivularis as a male parent, and using fertile individuals in the triploid induction group line of the ostrea rivularis as female parents to obtain a tetraploid rapid growth line of the ostrea rivularis through induction;
D. using the quick growth line of the near river oyster diploid as a male parent and using the quick growth line of the hong Kong oyster diploid as a female parent, and performing interspecific hybridization to obtain a hybrid oyster diploid quick growth line;
E. the tetraploid fast growing line of the crassostrea rivularis is taken as a male parent, and the diploid fast growing line of the hybridized crassostrea rivularis is taken as a female parent, and the hybridization is carried out to obtain the triploid fast growing new strain of the hong Kong oyster.
2. The method of claim 1, wherein the method for breeding a new triploid rapid-growth line of crassostrea hongkongensis by backcrossing comprises the following steps:
A. respectively collecting 3-5 different geographical group lines of the diploid of the crassostrea hongkongensis and the diploid of the crassostrea crassoensis, continuously breeding for more than 4 generations by taking growth and survival as target characters, and respectively breeding to obtain the rapid growth line of the diploid of the crassostrea hongkongensis and the rapid growth line of the diploid of the crassostrea crassoensis;
B. respectively taking the quick growth line of the ostrea rivularis diploid in the step A as a parent, and after 2N male parent and 2N female parent fertilization, using CB to inhibit pb2 release to obtain a ostrea rivularis triploid induction line;
C. taking the near river oyster diploid in the step A as a male parent, taking a fertile individual in the near river oyster triploid induction population in the step B as a female parent, and inhibiting the release of the polar body after fertilization by 2N male parent multiplied by 3N female parent to obtain a near river oyster tetraploid rapid growth line through induction;
D. taking the quick growth line of the near river oyster diploid in the step C as a male parent, taking the quick growth line of the hong Kong oyster diploid as a female parent, and obtaining a hybrid oyster diploid quick growth line after interspecific hybridization of 2N male parent and 2N female parent fertilization;
E. and (4) hybridizing the tetraploid rapid growth line of the ostrea rivularis in the step (C) with the diploid rapid growth line of the hybridized oysters in the step (D) as a female parent to obtain the triploid rapid growth new strain of the hong Kong oysters.
3. The method of claim 2, wherein the method for breeding the rapidly growing new line of the triploid crassostrea hongkongensis by using the backcross technology comprises the following specific steps:
A. breeding the diploid rapid growth line of the hong Kong oyster and the ostrea rivularis: collecting 3-5 different wild populations of Crassostrea hongkongensis and Crassostrea rivularis respectively, analyzing the interaction between genotype and environment type by taking growth and survival as targets, and screening out the populations with the best performance respectively; self-breeding the optimal population, continuously breeding 4 generations according to the seed reserving rate of 10 percent for each generation by taking growth and survival as breeding targets, and respectively breeding a Hongkong oyster diploid rapid growth line and a crassostrea gigas diploid rapid growth line;
B. constructing a crassostrea rivularis triploid induction line: taking individuals with good shell type and large full weight of the ostrea rivularis diploid rapid growth line in the A, arranging the individuals from large to small according to shell height, taking the first 10% of the individuals as parents, dissecting and identifying males and females after temporary culture, artificially fertilizing, inducing and treating by cytochalasin B when about 50% of a first polar body Pb1 is released after fertilization, and incubating and selecting excellent larvae, namely the ostrea rivularis triploid induction line;
C. constructing a tetraploid rapid growth line of the ostrea rivularis: and (4) respectively taking individuals with good shell type and large full weight of the ostrea rivularis diploid rapid growth line in the step a and the ostrea rivularis triploid induction line in the step b, and identifying the triploid ploidy after temporary culture. Respectively screening individuals with 10% shell height as parents; taking a diploid as a male parent and a triploid fertile female individual as a female parent, carrying out artificial fertilization, when about 50% of first polar body is released, using 6-dimethylaminopurine for induction, putting the first polar body into seawater for incubation, optimizing D-type larvae and detecting the tetraploid rate, and culturing and cultivating the first polar body in an excellent sea area, wherein the first polar body is a tetraploid induction line of the ostrea rivularis; screening individuals with larger overall weight and uniform size from the tetraploid induction line of the ostrea rivularis, identifying the tetraploid by a flow cytometer, arranging the tetraploid according to the shell height from large to small, taking the first 10 percent as a parent, carrying out artificial fertilization, selecting excellent larvae and identifying the tetraploid rate, thus obtaining the rapid growth line of the ostrea rivularis tetraploid;
D. constructing a rapid growth line of the diploid of the hybridized oyster: respectively taking individuals with large full weight and good shell shape of the near river oyster diploid fast growing line and the hong Kong oyster diploid fast growing line in the step a; after temporary rearing, arranging the shell heights from large to small, selecting the first 10% of individuals as parents, dissecting and identifying males and females, taking ostrea rivularis as a male parent and hong Kong oysters as a female parent, performing artificial fertilization, and breeding larvae, thus obtaining the hybrid oyster diploid rapid growth line;
E. cultivating a backcross triploid rapid growth line: and (4) respectively taking individuals with heavier weight and better shell shape of the tetraploid rapid growth line of the ostrea rivularis in the step (c) and the diploid rapid growth line of the hybrid oyster in the step (d), identifying the tetraploid after temporary rearing, carrying out artificial fertilization by using a tetraploid male and the diploid female of the hybrid oyster, hatching, selecting and identifying the triploid rate, and finely cultivating larvae and juvenile mollusks, which are the new strains for rapid growth of the triploid of the hong Kong oyster.
4. The method according to claim 3, wherein the different wild populations of Crassostrea hongkongensis and Crassostrea rivularis in step A are populations collected in the wild, with different geographical regions and high degree of variation.
5. The method as claimed in claim 3, wherein the constructing of the crassostrea rivularis triploid induction line, the ovum soaking, the fertilization and the larva cultivation seawater in the step B are carried out at the temperature of 29-31 ℃ and the salinity of 15ppt, the cytochalasin B induction concentration is 0.50mg/L, and after the fertilization, when about 50% of the Pb1 is released, the cytochalasin B induction treatment is carried out for 30 min.
6. The method as claimed in claim 3, wherein the rapid tetraploid growth line of Ostrea rivularis in step C is constructed by soaking ovum, fertilizing and incubating larva with seawater at 29-31 deg.C and 15ppt of salinity, and the inducing with 6-dimethylaminopurine is induced with 100mg/L of 6-DMAP for 65min when the first polar body is released about 50%.
7. The method of claim 3, wherein the larvae are refined in steps A, B, C, D and E, and the bait species are adjusted at different development stages, wherein the D-type larval stage bait is gold algae, the shell top larval stage bait is gold algae, cloud microalgae mixed bait, and the attached larvae are chaetoceros, Platymonas, and cloud microalgae mixed bait.
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