CN114600805A - Breeding method of 'Shibei No. 1' of rapid-growing triploid portuguese oyster - Google Patents
Breeding method of 'Shibei No. 1' of rapid-growing triploid portuguese oyster Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a method for cultivating 'Shibei No. 1' rapid-growing triploid portuguese oysters, and belongs to the technical field of aquaculture breeding. The invention provides a method for cultivating 'Shibei No. 1' rapid-growth triploid portuguese oysters, which comprises the following steps: selecting the crassostrea viticola with the weight of 100-; taking the F generation as a parent, inducing to obtain a triploid, and then taking the triploid as a parent, inducing to obtain a tetraploid G; self-propagating tetraploid G continuous groups to obtain tetraploid G' with stable ploidy; the tetraploid G ' is used as a male parent, the F generation is used as a female parent for hybridization, and the fast-growing triploid portuguese oyster ' Shibei No. 1 ' is obtained by cultivation. The Shimansu No. 1 cultivated by the method has the advantages of high growth speed, short cultivation period, strong stress resistance, excellent quality and the like, and has high popularization value.
Description
Technical Field
The invention belongs to the technical field of aquaculture breeding, and particularly relates to a culture method of 'Shibei No. 1' of rapid-growth triploid portuguese oysters.
Background
China is a world big oyster cultivation country, the oyster cultivation area of China is 16.49 hectares in 2020, the yield is 542.46 ten thousand tons, and the oyster cultivation area is at the top of the world. At present, the major breeding varieties of oysters in China are crassostrea gigas, crassostrea viticola and the like, wherein the breeding area and the yield of the crassostrea viticola are the largest, and the breeding main producing area is located in coastal areas such as Fujian and Guangdong.
In recent years, with the increasing demand for high-quality oysters, the breeding and breeding of triploid oysters has been developed vigorously. The ostrea aurantiaca has the advantages of high growth speed, short culture period and the like, but the ostrea aurantiaca is influenced by the reproduction activity and is easy to cause the emaciation of a soft body part, and the triploid ostrea is not influenced by the reproduction activity and keeps higher glycogen content and quality all the year round. Therefore, the method for culturing the portuguese oyster triploid with high growth speed, short culture period and good quality has important significance for promoting transformation and upgrade of oyster culture industry in China and assisting rural joy.
Disclosure of Invention
In view of the above, the invention aims to provide a cultivation method of the rapid-growing triploid portuguese oyster 'world times No. 1', which can be used for producing the rapid-growing triploid portuguese oyster 'world times No. 1' with high growth speed, strong stress resistance, excellent quality and obvious advantages in large scale.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for cultivating 'Shibei No. 1' rapid-growing triploid grapevine oysters, which comprises the following steps: selecting the crassostrea viticola with the weight of 100-; taking the F generation as a parent, inducing to obtain a triploid, and then taking the triploid as a parent, inducing to obtain a tetraploid G; self-propagating tetraploid G continuous groups to obtain tetraploid G' with stable ploidy; the tetraploid G ' is used as a male parent, the F generation is used as a female parent for hybridization, and the fast-growing triploid portuguese oyster ' Shibei No. 1 ' is obtained by cultivation.
Preferably, the ostrea aurantiaca is ostrea aurantiaca of 2-3 ages.
Preferably, the generation number of the directional breeding is more than 4.
Preferably, the induction method is to treat the fertilized egg with cytochalasin B.
Preferably, the concentration of the cytochalasin B is 0.4-0.6 mg/L.
Preferably, the treatment time of the cytochalasin B is 20-40 min.
Preferably, after the fertilized egg is treated with cytochalasin B, the fertilized egg is soaked in a dimethyl sulfoxide solution.
Preferably, the concentration of the dimethyl sulfoxide solution is 1%, and the soaking time is 15 min.
Preferably, the induced fertilized egg is subjected to flow cytometric screening after the induction.
Preferably, the number of generations of the continuous population is 2-3 generations.
The invention has the beneficial effects that:
the invention takes the shell height and the weight as target characters, adopts a method combining group breeding with polyploid breeding to develop the rapid-growing line breeding of the portuguese oyster, then uses the rapid-growing line breeding to induce the portuguese oyster tetraploid and carries out the group self-breeding, and finally crosses the progeny of the rapid-growing line diploid and the tetraploid self-breeding, thereby cultivating the rapid-growing portuguese oyster triploid 'Shimanshu No. 1'. The 'Shibei No. 1' cultivated by the method has the advantages of high growth speed, strong stress resistance, excellent quality and the like, is very suitable for coastal cultivation in China, has wide popularization and application prospects, and is beneficial to improving the popularity of triploid oysters in high-end consumption markets, so that the transformation and upgrade of oyster industry in China to the high-end consumption markets are promoted, and the sustainable healthy green development of oyster cultivation industry in China is promoted.
Drawings
FIG. 1 is a technical route chart of the 'Shibei No. 1' cultivation method of the rapid-growth molar oyster triploid of the invention;
FIG. 2 shows the growth of the sea area "Shi Bei No. 1";
FIG. 3 is a detection peak diagram of a rapid-growth portuguese oyster triploid individual;
FIG. 4 is a graph of tetraploid induction peaks of Ostrea gigas Thunb.
Detailed Description
The invention provides a method for cultivating 'Shibei No. 1' rapid-growing triploid grapevine oysters, which comprises the following steps: selecting the crassostrea viticola with the weight of 100-; taking the F generation as a parent, inducing to obtain a triploid, and then taking the triploid as a parent, inducing to obtain a tetraploid G; self-propagating tetraploid G continuous groups to obtain tetraploid G' with stable ploidy; the tetraploid G ' is used as a male parent, the F generation is used as a female parent for hybridization, and the fast-growing triploid portuguese oyster ' Shibei No. 1 ' is obtained by cultivation.
The specific sources of the crassostrea viticola in the present invention are not particularly limited, and include cultivated crassostrea and wild crassostrea, and in the present invention, the crassostrea viticola is preferably crassostrea viticola of 2 to 3 years old, and more preferably crassostrea viticola of 2 years old. When the method is used for screening the Ostrea clarkii parents, the weight and the shell height are taken as target properties, wherein the weight is preferably 140 g-120 g, more preferably 135 g-130 g, and the shell height is preferably 10-11 cm. In the invention, the directional breeding method preferably collects sperms and ova of selected breeding parents, obtains D-type larvae by adopting an artificial insemination method, cultures the D-type larvae to eyespot larvae in an indoor culture pond, puts in an attachment group, moves oyster juvenile mollusks growing until the shell height is 2-3mm to a natural sea area for culturing until the oysters become shells, screens again by taking the shell height and the body weight as target characters, and the directional breeding is carried out continuously. In the invention, the generation number of the directional breeding is preferably more than 4 generations, more preferably 6-8 generations, and the rapid breeding line F generation of the portuguese oyster is obtained.
In the present invention, the F generation is used as a parent to induce triploid, the induction method is preferably to treat the fertilized egg with cytochalasin B, the concentration of cytochalasin B is preferably 0.4-0.6mg/L, more preferably 0.5mg/L, the treatment time of cytochalasin B is preferably 20-40min, more preferably 30min, and the specific source of cytochalasin B is not particularly limited in the present invention. In the present invention, the method for treating a fertilized egg with cytochalasin B is preferably: dissecting female and male shellfish of F generation of sex gland mature breeding system, collecting sperm and ovum, artificial insemination, and treating with cytochalasin B when fertilized ovum grows to the first polar body. The ratio of the number of the ovum and the number of the sperm in the artificial insemination is preferably 1:5-10, more preferably 1:6-8, and most preferably 1: 7. After the fertilized eggs are treated by cytochalasin B, the fertilized eggs are preferably soaked in dimethyl sulfoxide solution, and then conventional hatching, larva cultivation and sea area cultivation are carried out. The concentration of the dimethyl sulfoxide solution is preferably 1%, and the soaking time is preferably 15 min.
In the invention, when the triploid is used as a parent and tetraploid G is obtained through induction, the fertilized egg is preferably obtained by artificial insemination by using the rapid-growth portuguese oyster triploid as a female parent and using a rapid-growth breeding line F diploid as a male parent. The method for inducing the rapid portuguese oyster tetraploid G is the same as the method for inducing the rapid portuguese oyster triploid, and the operation steps are not repeated herein.
In the present invention, after the induction, it is preferable to further include performing flow cytometry screening on the fertilized eggs after the induction to obtain a triploid of rapid portuguese oyster with a ploidy of 3N and a tetraploid G of rapid portuguese oyster with a ploidy of 4N.
After obtaining the tetraploid G, the invention carries out self-reproduction on the tetraploid G continuous population, the self-reproduction generation number of the continuous population is preferably 2-3, and the tetraploid G' with stable ploidy is obtained. In the invention, the self-breeding method of the population preferably comprises the steps of respectively collecting ova and sperms in female and male shells of the tetraploid G of the rapid grapevine oyster, carrying out artificial insemination, adjusting density for cultivation when obtained fertilized ova grow to D-type larvae, and putting attaching bases when the larvae grow to eyepoints; when the growth shell of the young shellfish is 2-3mm high, putting the young shellfish into an upflow system for cultivation; when the height of the young shellfish growing shell is more than 1cm, removing the base, and obtaining the monomer oyster which is filled into a cage and moved to a natural sea area for cultivation. The breeding density of the D-type larvae is preferably 1-2/ml. The attaching base preferably comprises oyster shells, scallop shells, plastic sheets, cement blocks, cement strips and cement cakes.
After the tetraploid G 'with stable ploidy is obtained, the tetraploid G' is taken as a male parent, the F generation is taken as a female parent for hybridization, and the fast-growing triploid portuguese oyster 'Shibei No. 1' is obtained by cultivation. The method of hybridization according to the present invention is not particularly limited, and in vitro hybridization methods well known in the art may be used.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
And (3) breeding a rapid growth breeding line F of the portuguese oysters: 20000 oyster culture and wild 2-3-age groups are collected from coastal areas of Fujian and Zhejiang, 1000 individuals with the shell height and the weight of the former 5% are taken as parent breeding scallops, the weight of the parent breeding scallops is 150g, the shell shape is neat, and the shell height is 10-12 cm. Obtaining D-type larvae by artificial insemination, culturing in an indoor cement pond until eyespot larvae grow, and putting oyster shells as an attaching base; when the young oyster grows to 2-3mm, moving to a natural sea area for cultivation. Finally, the rapid-growing breeding line F6 of the portuguese oyster is bred by continuous 6 generations of population selection according to the same breeding target, breeding technology and breeding method.
Induction of rapid-growing grapevine oyster tetraploid G: inducing triploid: dissecting 100 rapid breeding lines F6 parents of the mature grapevine oysters with individual glands, and separating out male and female (28) parents (32) by using a microscope; extruding 20 pairs of male and female with good gonads to obtain fine eggs, respectively filtering the fine eggs and the fine eggs by a 200-mesh silk screen and then by a 500-mesh silk screen; adopting a single-pair single method to fertilize, and simultaneously controlling 5-10 sperms around each ovum; when 10% of fertilized eggs develop to the first polar body, 0.5mg/L cytochalasin B is immediately added to treat the fertilized eggs for 30min, and then the fertilized eggs are soaked in seawater containing 1% of dimethyl sulfoxide for 15 min; collecting fertilized eggs of each group, putting the fertilized eggs into an indoor cement pond for hatching and larva cultivation, detecting the triploid induction rate to be more than 90 percent by using a flow cytometer, and finally culturing the fertilized eggs in a sea area. Induction of tetraploid: dissecting gonad mature rapid portuguese oyster triploid, detecting 100 individuals with ploidy of 3N by using a flow cytometer, and screening 30 female 3N individuals with well developed ova as female parents by observing through a microscope; dissecting 100 rapid propagation breeding lines F6 parents of mature grapevine oysters with individual glands, distinguishing sexes by using a microscope, and selecting 30 individuals with plump gonads and strong sperm motility as male parents; respectively extruding out essence and ovum, filtering with a 200-mesh silk screen, and then filtering with a 500-mesh silk screen; adopting a single-pair single method to fertilize, and simultaneously controlling 5-10 sperms around each ovum; when 50% of fertilized eggs develop to the first polar body, 0.5mg/L cytochalasin B is immediately added to treat the fertilized eggs for 30min, and then the fertilized eggs are soaked in seawater containing 1% of dimethyl sulfoxide for 15 min; collecting fertilized eggs of each group, putting the fertilized eggs into an indoor cement pond for hatching and larva cultivation, detecting that the tetraploid induction rate is more than 40 percent by using a flow cytometer, and finally cultivating 10000 rapid-growing grapevine oyster tetraploids in a sea area.
Self-reproduction of the rapid grapevine oyster tetraploid population G: in the oyster breeding season, taking the rapid-growing grapevine oyster tetraploid G as a basic population, and screening 100 individuals with ploidy of 4N males and females by a flow cytometer; respectively extruding out essence and ovum, filtering with a 200-mesh silk screen, and then filtering with a 500-mesh silk screen; controlling 3-5 sperms around each ovum in the artificial insemination process; when fertilized eggs develop into D-type larvae, collecting the larvae in the whole pond, and putting the larvae into a new sterilized cement pond for cultivation, wherein the larva cultivation density is 1-2 larvae per ml; when the larva grows to the eyespot, putting a polyethylene plastic shell as an attaching base; when the juvenile mollusks grow to 2-3mm, dispersing polyethylene plastic sheets and putting the polyethylene plastic sheets into an upflow system for cultivation; when the juvenile mollusks grow to be more than 1cm, monomer oysters are obtained by a basal removal method and are filled in a cage and moved to a natural sea area for cultivation. The same method is adopted to carry out the self-reproduction of the tetraploid population of the continuous 2 generations, thereby producing the rapid-growing grapevine oyster tetraploid G2 with stable ploidy.
Producing rapid-growing Portugal oyster triploid 'Shidoubly No. 1': transferring 1000 rapid-growing breeding lines of the grapevine oysters F6 individuals and 100 rapid-growing grapevine oyster tetraploid G2 individuals to an indoor cement pond for constant-temperature (23 ℃) ripening, wherein the biological bait is artificially cultured chrysophyceae, chaetoceros, diatom and tetraselmis; after the gonads are mature, 90 male individuals of the rapid-growth grapevine oyster tetraploid G2 are obtained through flow cytometry detection and microscope observation for later use; dissecting 1000 individuals of the rapid growth breeding line F6 of the portuguese oyster, and selecting 600 female individuals for later use by microscope observation; hybridizing with G2 male individual as male parent and F6 female individual as female parent, and extruding sperm and egg from the parentsFiltering with 200 mesh silk screen and then with 500 mesh silk screen; controlling 3-5 sperms around each ovum in the artificial insemination process; after fertilization, fertilized eggs were divided into 20 mouths and 30m2Hatching in a cement pond; when the larvae develop to D type, 2000g (about 100 hundred million) of larvae are collected by a 400-mesh silk net; evenly dividing D-type larvae into 120 sterilized 30m2Larva breeding is carried out in a cement pond, and the breeding density is controlled to be 1-3/ml; when the larva grows to the eyespot larva, oyster shells are put in, and the metamorphosis rate of the larva is more than 30%; when the juvenile mollusks grow to 2-3mm, moving to a natural sea area for temporary culture; after temporary culture for 15 days in the sea area, artificial seedling separation is carried out, and then the seedlings are moved to a natural sea area for culture. The technical route of the breeding method is shown in figure 1.
After 1 year cultivation in the sea area, the growth of the "Shibei No. 1" sea area is shown in FIG. 2. The average total weight of 'Shibei No. 1' is up to 95.25g, which is improved by 20.55 percent compared with the control group (common portuguese oyster triploid); the average shell height of 'Shibei No. 1' is 12.98cm, which is improved by 17.47% compared with the control group (common portuguese oyster triploid); the average shell length of 'Shibei No. 1' is 6.75cm, which is improved by 3.53 percent compared with a control group (common portuguese oyster triploid); the average shell width of 'Shibei No. 1' is 4.63cm, which is 3.81% higher than that of the control group (ordinary portuguese oyster triploid), as shown in Table 1.
TABLE 1 comparison of the growth of "Shibei No. 1" cultivated in 1 year with the growth of the sea area of the control group
Example 2
And (3) breeding a rapid growth breeding line F of the portuguese oysters: 10000 oyster culture and wild population of 3 years are collected from the coastal region of Guangdong, and then 500 individuals with the shell height and the weight which is 5 percent of the weight of the parent oyster are taken as parent oysters for propagation, wherein the weight of the parent oysters for propagation is 100-130g, the shell shapes are regular, and the shell height is 9-11 cm. Obtaining D-type larvae by artificial insemination, culturing in an indoor cement pond until eyespot larvae grow, and putting oyster shells as an attaching base; when the young oyster grows to 2-3mm, moving to a natural sea area for cultivation. Finally, the rapid-growing breeding line F7 of the portuguese oyster is bred through continuous 7-generation population selection by the same breeding target, breeding technology, breeding method and the like.
Induction of rapid-growing grapevine oyster tetraploid G: inducing triploid: dissecting 100 rapid breeding lines F7 parents of the mature grapevine oysters with individual glands, and separating 40 males and females (45) by using a microscope; extruding 30 pairs of male and female with good gonads to obtain fine eggs, respectively filtering the fine eggs and the fine eggs by a 200-mesh silk screen and then by a 500-mesh silk screen; adopting a single-pair single method to fertilize, and simultaneously controlling 5-10 sperms around each ovum; when 10% of fertilized eggs develop to the first polar body, 0.5mg/L cytochalasin B is immediately added to treat the fertilized eggs for 30min, and then the fertilized eggs are soaked in seawater containing 1% of dimethyl sulfoxide for 15 min; collecting fertilized eggs of each group, putting the fertilized eggs into an indoor cement pond for hatching and larva cultivation, detecting the triploid induction rate to be more than 90 percent by using a flow cytometer, and finally culturing the fertilized eggs in a sea area. The detection profile of triploid individuals is shown in fig. 3. Induction of tetraploid: dissecting gonad mature rapid portuguese oyster triploid, detecting 100 individuals with ploidy of 3N by using a flow cytometer, and screening 40 female 3N individuals with well developed ova as female parents by observing through a microscope; dissecting 100 rapid propagation breeding lines F7 parents of mature grapevine oysters with individual glands, distinguishing sexes by using a microscope, and selecting 40 individuals with plump gonads and strong sperm motility as male parents; respectively extruding out essence and ovum, filtering with a 200-mesh silk screen, and then filtering with a 500-mesh silk screen; adopting a single-pair single method to fertilize, and simultaneously controlling 5-10 sperms around each ovum; when 50% of fertilized eggs develop into the first polar body, 0.5mg/L cytochalasin B is immediately added to treat the fertilized eggs for 30min, and then the fertilized eggs are soaked in seawater (containing 1% of dimethyl sulfoxide) for 15 min; collecting fertilized eggs of each group, placing the fertilized eggs into an indoor cement pond for hatching and larva cultivation, and detecting the tetraploid induction rate to be more than 40% by using a flow cytometer, as shown in figure 4. Finally, about 8000 rapid-growing portuguese oyster tetraploids are cultivated in the sea area.
Self-reproduction of the rapid grapevine oyster tetraploid population G: in the oyster breeding season, taking the rapid-growing grapevine oyster tetraploid G as a basic population, and screening 100 individuals with ploidy of 4N males and females by a flow cytometer; respectively extruding out essence and ovum, filtering with a 200-mesh silk screen, and then filtering with a 500-mesh silk screen; in the artificial insemination process, controlling 3-5 sperms around each ovum; when fertilized eggs develop into D-type larvae, collecting the larvae in the whole pond, and putting the larvae into a new sterilized cement pond for cultivation, wherein the larva cultivation density is 1-2 larvae per ml; when the larva grows to the eyespot, putting a polyethylene plastic shell as an attaching base; when the juvenile mollusks grow to 2-3mm, dispersing polyethylene plastic sheets and putting the polyethylene plastic sheets into an upflow system for cultivation; when the juvenile mollusks grow to be more than 1cm, monomer oysters are obtained by a basal removal method and are filled in a cage and moved to a natural sea area for cultivation. The same method is adopted to carry out the self-reproduction of the tetraploid population of the continuous 3 generations, thereby producing the rapid-growing grapevine oyster tetraploid G3 with stable ploidy.
Producing rapid-growing Portugal oyster triploid 'Shidoubly No. 1': 3000 rapid-growing breeding lines of the portuguese oysters F7 individuals and 300 rapid-growing portuguese oyster tetraploid G3 individuals are moved to an indoor cement pond to be subjected to constant-temperature (23 ℃) ripening acceleration, and biological baits are artificially cultured golden algae, chaetoceros, diatom and platymysternon; after the gonads are mature, 136 male individuals of the rapid-growth grapevine oyster tetraploid G2 are obtained through flow cytometry detection and microscope observation for later use; dissecting 3000 rapid breeding lines of the portuguese oysters F7 individuals, and selecting 1800 female individuals for later use by microscope observation; hybridizing a G3 male individual serving as a male parent and a F7 female individual serving as a female parent, specifically extruding sperm and eggs from the parents, and filtering the sperm and the eggs by using a 200-mesh silk net and then a 500-mesh silk net respectively; controlling 3-5 sperms around each ovum in the artificial insemination process; after fertilization, fertilized eggs were divided into 30 mouths and 30m2Hatching in a cement pond; when the larvae develop to D type, 3000g (about 150 hundred million) of larvae are collected by a 400-mesh silk net; dividing D type larva into 200 sterilized 30m2Larva breeding is carried out in a cement pond, and the breeding density is controlled to be 1-3/ml; when the larva grows to the eyespot larva, oyster shells are put in, and the metamorphosis rate of the larva is more than 30%; when the juvenile mollusks grow to 2-3mm, moving to a natural sea area for temporary culture; after temporary culture for 15 days, artificial seedling separation is carried out, and then the seedlings are moved to natural sea areas for culture.
After 1 year of cultivation in the sea area, the average total weight of 'Shibei No. 1' reaches 106.34g, which is improved by 29.43 percent compared with that of a control group (ordinary portuguese oyster triploid); the average shell height of 'Shibei No. 1' is 13.08cm, which is improved by 19.28 percent compared with the control group (common portuguese oyster triploid); the average shell length of 'Shibei No. 1' is 7.37cm, which is 10.33 percent higher than that of a control group (common portuguese oyster triploid); the average shell width of 'Shibei No. 1' was 4.94cm, which was 9.05% higher than that of the control group (ordinary Portuguese oyster triploid), and the results are shown in Table 2.
TABLE 2 comparison of the growth of "Shibei No. 1" cultivated in 1 year with the growth of the sea area of the control group
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A cultivation method of 'Shibei No. 1' of rapid-growth triploid portuguese oysters is characterized by comprising the following steps: selecting the crassostrea viticola with the weight of 100-; taking the F generation as a parent, inducing to obtain a triploid, and then taking the triploid as a parent, inducing to obtain a tetraploid G; self-propagating tetraploid G continuous groups to obtain tetraploid G' with stable ploidy; the tetraploid G ' is used as a male parent, the F generation is used as a female parent for hybridization, and the fast-growing triploid portuguese oyster ' Shibei No. 1 ' is obtained by cultivation.
2. The method of claim 1, wherein the oysters of portugal oyster are 2-3 instar oysters of portugal oyster.
3. The method of claim 1, wherein the generation number of the targeted breeding is more than 4.
4. The method of claim 1, wherein the inducing is performed by treating the fertilized egg with cytochalasin B.
5. The method of claim 4, wherein said cytochalasin B is present at a concentration of 0.4-0.6 mg/L.
6. The method of claim 4, wherein said cytochalasin B treatment is for a period of 20-40 min.
7. The method according to claim 4, wherein the fertilized egg is soaked with a dimethylsulfoxide solution after the fertilized egg is treated with cytochalasin B.
8. The method according to claim 7, wherein the concentration of the dimethyl sulfoxide solution is 1%, and the soaking time is 15 min.
9. The method of claim 1, wherein the induced zygotes are subjected to flow cytometric screening following induction.
10. The method of claim 1, wherein the number of generations of the self-reproduction of the continuous population is 2-3 generations.
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