CN107027661B - Hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and production method thereof - Google Patents
Hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and production method thereof Download PDFInfo
- Publication number
- CN107027661B CN107027661B CN201611196783.0A CN201611196783A CN107027661B CN 107027661 B CN107027661 B CN 107027661B CN 201611196783 A CN201611196783 A CN 201611196783A CN 107027661 B CN107027661 B CN 107027661B
- Authority
- CN
- China
- Prior art keywords
- epinephelus
- hybrid
- fuscoguttatus
- lanceolatus
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241001217936 Epinephelus lanceolatus Species 0.000 title claims abstract description 58
- 241001149251 Epinephelus fuscoguttatus Species 0.000 title claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 title abstract description 17
- 241000251468 Actinopterygii Species 0.000 claims abstract description 23
- 230000001418 larval effect Effects 0.000 claims abstract description 16
- 235000013601 eggs Nutrition 0.000 claims description 43
- 210000000582 semen Anatomy 0.000 claims description 18
- 102000053602 DNA Human genes 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 17
- 238000001962 electrophoresis Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 12
- 238000003752 polymerase chain reaction Methods 0.000 claims description 10
- 230000004720 fertilization Effects 0.000 claims description 9
- 230000012447 hatching Effects 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 238000009395 breeding Methods 0.000 abstract description 14
- 230000001488 breeding effect Effects 0.000 abstract description 14
- 238000009396 hybridization Methods 0.000 abstract description 8
- 230000004913 activation Effects 0.000 abstract description 3
- 235000019688 fish Nutrition 0.000 description 16
- 241000894007 species Species 0.000 description 14
- 241001417495 Serranidae Species 0.000 description 8
- 241000357439 Epinephelus Species 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000357444 Epinephelus coioides Species 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 4
- 239000013535 sea water Substances 0.000 description 4
- 241001247197 Cephalocarida Species 0.000 description 3
- 241000788447 Epinephelus aeneus Species 0.000 description 3
- 241000321397 Epinephelus analogus Species 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000384 rearing effect Effects 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 241001596774 Epinephelus akaara Species 0.000 description 2
- 241000950696 Epinephelus chlorostigma Species 0.000 description 2
- 108700012941 GNRH1 Proteins 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 101000640713 Homo sapiens Transmembrane protein 126A Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000700141 Rotifera Species 0.000 description 2
- 102100033846 Transmembrane protein 126A Human genes 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 208000009606 optic atrophy 7 Diseases 0.000 description 2
- 208000012242 optic atrophy 8 Diseases 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000448486 Epinephelinae Species 0.000 description 1
- 241000980405 Epinephelus costae Species 0.000 description 1
- 241001098499 Lanceolata Species 0.000 description 1
- 206010037867 Rash macular Diseases 0.000 description 1
- 241000269821 Scombridae Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 235000020640 mackerel Nutrition 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Organic Chemistry (AREA)
- Marine Sciences & Fisheries (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to a hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and a production method thereof, in particular to a hybrid produced by hybridization between Epinephelus fuscoguttatus and Epinephelus lanceolatus belonging to the subfamily Epinephelus lanceolatus in the bass family and a production method of the hybrid thereof, and relates to a hybrid between female Epinephelus fuscoguttatus and male Epinephelus lanceolatus and a production method of a hybrid between male Epinephelus fuscoguttatus and female Epinephelus lanceolatus. The invention can provide hybrid seeds with high growth speed and excellent quality through hybridization between the high marketability Epinephelus fuscoguttatus and the fast-growing and marketable Epinephelus lanceolatus. The hybrid of the present invention has a fast growth of the initial larval fish, and thus solves the problem of biological bait, which is a major problem when breeding bass, and contributes to the activation of the korean breeding industry.
Description
Technical Field
The present invention relates to a hybrid produced by crossing two kinds of Epinephelus coioides and Epinephelus lanceolatus belonging to the subfamily Epinephedae of the family Sermonidae and a method for producing the hybrid thereof, and relates to a hybrid between female Epinephelus lanceolatus and male Epinephelus lanceolatus and a method for producing the hybrid between male Epinephelus lanceolatus and female Epinephelus lanceolatus.
The invention is a method comprising the steps of: comprises the steps of ensuring mature eggs from females, carrying out artificial fertilization between the two after extracting semen from males so as to produce fertilized eggs, and breeding the fertilized eggs into hatchlings and seedlings.
Compared with Epinephelus fuscoguttatus, the hybrid between Epinephelus fuscoguttatus and Epinephelus lanceolatus has the advantage of very fast growth.
Background
The species of bass (family Serranidae) are 529 species of the 3 subfamilies, of which the major species of breeding is the subfamily Epinephelinae (subfamily Epinepineae), are the 15 species of the genus 159, and most inhabit in the Indian-Pacific region, which is subtropical.
The bass attracts attention as a high-priced breeding fish, and the breeding production amount is rapidly increased, i.e., 94040 tons is produced in 2011, thereby having a large marketability which is increased by 7.3 times in 10 years.
The mackerel, which is the species subject to the present invention, is sold at high prices as a fish species having high added value in the aquaculture industry, called "eastern bass" in the dialect of china, and is in great demand in the hong kong market and the native china, which are the largest markets for bass.
Epinephelus lanceolata, an ultra-large fish living in subtropical regions, attracts attention as a breeding target species in Taiwan due to its rapid growth rate and high price, and is also gradually introduced into China.
Hybridization is a technique for producing a new species for growth, disease resistance, meat quality, environmental fitness, and the like, which are economically important traits, as a mating method between two types of different genotypes and phenotypes.
In particular, interspecific crossing has a high probability of acquiring heterosis through combination of desired traits, and thus many attempts are being made (Korean laid-open patent No. 10-1999-0045938, Korean granted patent No. 10-0844339, Korean laid-open patent No. 10-2012-0083990).
On the other hand, production of various hybrid varieties targeted for bass is also actively being performed.
Typically, there are dark spot groupers (black-fin groupers (e.aeneus)), white groupers (gold groupers (e.costae)), dark spot groupers (brown-padded groupers (e.fuscogdates)), white groupers (spotted groupers (e.photoskadion)), spotted groupers (orange-spotted groupers (e.coioides)), brown-spotted groupers (brown-spotted groupers), spotted groupers (e.akaaria), and red-spotted groupers (e.akaara)), etc.
In particular, hybrid seeds obtained by artificial fertilization of mature eggs of female Epinephelus coioides and semen of Epinephelus lanceolatus have rapid growth and disease resistance, are mass-produced, and are registered and sold in the hong Kong market.
However, hybridization of bass has only been a hybrid between subtropical species to date, and hybridization between bass and subtropical bass is not achieved in korea.
Meanwhile, when a new species is developed by crossing the high marketability blotchy grouper with the fast-growing and marketable epinephelus lanceolatus, it can be sold in china having the largest consumer market together with the sale in korea.
Documents of the prior art
Patent document
Patent document 0001: korean laid-open patent No. 10-1999-0045938
Patent document 0002: korean granted patent No. 10-0844339
Patent document 0003: korean laid-open patent No. 10-2012 and 0083990
Disclosure of Invention
The present invention aims to develop a new species by hybridization in order to obtain excellent qualities such as marketability, growth rate and the like of Epinephelus fuscoguttatus and Epinephelus lanceolatus.
Further, an object of the present invention is to provide a method for hybridizing a limonitic grouper and a epinephelus lanceolatus, which has a high growth rate and can contribute to the activation of the breeding industry.
In order to achieve the above objects, the present invention provides a method for producing a hybrid of epinephelus fuscoguttatus and epinephelus lanceolatus, comprising: preparing mature eggs of female Epinephelus fuscoguttatus, and after preparing semen of male Epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of male Epinephelus fuscoguttatus to produce fertilized eggs; and a step of incubating and raising the fertilized eggs to produce larval fish.
The present invention also provides a method for producing a hybrid of epinephelus fuscoguttatus and epinephelus lanceolatus, comprising: preparing mature eggs of female epinephelus lanceolatus, and after preparing semen of male epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of the male epinephelus lanceolatus to produce fertilized eggs; and a step of incubating and raising the fertilized eggs to produce larval fish.
The present invention also provides a method for confirming a hybrid of epinephelus fuscoguttatus and epinephelus lanceolatus, the method comprising: extracting deoxyribonucleic acid (DNA) of the produced larval fish; mixing the extracted deoxyribonucleic acid of the larval fish with a primer of a sequence 1 or a primer of a sequence 2, and amplifying by using a Polymerase Chain Reaction (PCR); and a step of confirming a product obtained by the amplification by electrophoresis.
In one embodiment of the present invention, in the step of confirming by electrophoresis, when the primer of sequence 1 is used, if bands of about 320bp, 330bp, 400bp and 450bp are detected, it is determined as a hybrid.
In one embodiment of the present invention, in the step of confirming by electrophoresis, when the primer of sequence 2 is used, if bands of about 380bp and 410bp are detected, it is determined as a hybrid.
The present invention also provides a hybrid, fertilized egg and fry prepared by the method for producing the hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus.
The invention can provide hybrid seeds with high growth speed and excellent quality through hybridization between the high marketability Epinephelus fuscoguttatus and the fast-growing and marketable Epinephelus lanceolatus.
The hybrid of the present invention has a very rapid growth of the initial larval fish, and thus solves the problem of biological bait, which is a major problem when breeding bass, and contributes to the activation of the korean breeding industry.
Also, the present invention can improve the national competitiveness of the korean breeding industry against taiwan, the most developed country for the production of bass seedlings, and can create a large income by entering the bass breeding market.
Drawings
FIG. 1 shows the water temperature hatchability and initial survival rate of hybrid species of Epinephelus fuscoguttatus and Epinephelus lanceolatus.
FIG. 2 shows morphological characteristics of Epinephelus fuscoguttatus, Epinephelus lanceolatus and hybrids.
FIG. 3 shows the growth rate differences between Epinephelus fuscoguttatus and hybrids, using the exponential trend line, and disclosing the R2The value is obtained.
FIG. 4 is an electrophoretogram of PCR amplification products using primers of SEQ ID No. 1, wherein GG1, GG2 and GG3 are Epinephelus lanceolatus, LG1, LG2 and LG3 are Epinephelus lanceolatus, and LG XGG 1 and LG XGG 2 are hybrids.
FIG. 5 is an electrophoretogram of PCR amplification products using primers of SEQ ID No. 2, wherein GG1, GG2 and GG3 are Epinephelus lanceolatus, LG1, LG2 and LG3 are Epinephelus lanceolatus, and LG XGG 1 and LG XGG 2 are hybrids.
Detailed Description
The present invention will be described in detail below with reference to examples and the accompanying drawings. Terms, embodiments, drawings, and the like used in the present invention are only for more specifically illustrating the present invention and help those of ordinary skill in the art to understand the present invention, and the scope of the invention claimed and the like should not be construed as being limited thereto.
Technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs, unless defined otherwise.
The invention relates to a production method of hybrid seeds of Epinephelus fuscoguttatus and Epinephelus lanceolatus, which comprises the following steps: preparing mature eggs of female Epinephelus fuscoguttatus, and after preparing semen of male Epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of male Epinephelus fuscoguttatus to produce fertilized eggs; and a step of incubating and raising the fertilized eggs to produce larval fish.
Furthermore, the present invention relates to a method for producing hybrid seeds of Epinephelus fuscoguttatus and Epinephelus lanceolatus, comprising: preparing mature eggs of female epinephelus lanceolatus, and after preparing semen of male epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of the male epinephelus lanceolatus to produce fertilized eggs; and a step of incubating and raising the fertilized eggs to produce larval fish.
The invention also relates to a method for confirming the hybrid of the epinephelus fuscoguttatus and the epinephelus lanceolatus, which comprises the following steps: extracting the produced deoxyribonucleic acid of the larval fish; mixing the extracted deoxyribonucleic acid of the larval fish with a primer with a sequence 1 or a primer with a sequence 2, and amplifying by utilizing a polymerase chain reaction; and a step of confirming a product obtained by the amplification by electrophoresis.
In the step of confirming by electrophoresis, when the primer of sequence 1 is used, if bands of about 320bp, 330bp, 400bp and 450bp are detected, it is judged as a hybrid.
In the step of confirmation by electrophoresis, when the primer of sequence 2 is used, if bands of about 380bp and 410bp are detected, it is determined as a hybrid.
The present invention also relates to a hybrid, fertilized egg and fry prepared by the method for producing the hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus.
The present invention will be described in detail below with reference to examples. The following examples are only for carrying out the present invention, and the contents of the present invention are not limited to the following examples.
EXAMPLE 1 ensuring of parent Fish
In the present invention, as the Epinephelus fuscoguttatus, female Epinephelus fuscoguttatus, which is collected near Korea and matures in an individual having a weight of 1.5 to 5.2kg, which is domesticated in a parent fish rearing trough, is used, and as the Epinephelus lanceolatus, male Epinephelus lanceuttatus, which is imported from Taiwan and matures in an individual having a weight of 54.2 to 112.0kg, which is domesticated in a parent fish rearing trough, is used.
Example 2 hybridization
Eggs and semen were extracted from mature female Epinephelus fuscoguttatus and male Epinephelus lanceolatus, respectively, by abdominal compression, and then hybridized by artificial fertilization using a dry method.
First, in order to obtain mature eggs of mature female Epinephelus fuscoguttatus with high quality, the abdomen was observed, and the state of maturation was confirmed by touching with a hand.
Intraperitoneal injections of 25 μ g/kg of Luteinizing Hormone Releasing Hormone (LHRH) (Sigma, USA) and 1000 IU/kg of Human Chorionic Gonadotropin (HCG) were administered to the abdominal cavity of female adult limonitids.
The mature eggs were checked and confirmed by hand at intervals of 4 hours from 36 hours after the injection, and finally, the mature eggs were secured by pressing the abdomen.
Semen of male Epinephelus lanceolatus is extracted by abdominal compression.
The secured mature eggs are prevented from being exposed for more than 30 minutes at normal temperature, and after the secured mature eggs and semen are uniformly mixed in an egg collecting container, the mixture is allowed to stand for 1 to 5 minutes at room temperature, and then seawater is added to activate sperm.
Then, adding seawater, and standing for 5-20 minutes at room temperature, and then distinguishing floating eggs from sinking eggs.
After the floating eggs are moved to an egg washing net to wash the residual semen, the egg washing net floats in a water tank to flow out seawater for about 10-20 hours.
Finally, only the floating fertilized eggs are collected to move to a hatching tank.
Example 3 hatching and rearing
Hatching and raising of fish larvae were performed in the same manner in the hatching tank.
The results of incubation experiments showed that all incubation took place at the water temperatures of the incubation tanks up to 22-31 ℃ but the incubation rates at 22 ℃ and 31 ℃ were less than 25 ℃ and 28 ℃.
The survival rate of the yolk-absorbed fries was high in the 25 ℃ and 28 ℃ test groups, but did not survive in the 22 ℃ and 31 ℃ test groups (fig. 1).
The result of the aberration rate analysis of the hatched fries shows that at the temperature of 25 ℃, the aberration rate is 1.5 percent, and at the temperature of 28 ℃, the aberration rate is as high as 15.5 percent, so that the temperature of a proper hatching tank is preferably 23-27 ℃, more preferably 23.5-26.5 ℃.
Rotifers (rotifer) were supplied starting 2 days after the hatch.
As the fish larvae grow, the compound feed is supplied together with the artemia larvae. The breeding water is maintained exponentially in the initial stage of breeding by using filtered seawater, and the water change amount is maintained to be 30-50% in 1 day after the artemia are supplied. The excrement in the water tank is removed by a siphon tube from the artemia supplying step.
Example 4 investigation of growth Rate
The size of the fry after hatching, the size of the fry after yolk absorption, and the growth rate at intervals of 3 days, 5 days, 7 days, 12 days, 17 days, and 24 days after hatching were examined (fig. 2).
The measurements were carried out on the basis of the full length and, in order to compare the growth of the corresponding hybrids, on the basis of the full length of the pure species of the Epinephelus fuscoguttatus.
Until 20 days after incubation, there was no significant difference between the purebred limonite and the hybrid of limonite and epinephelus lanceolatus, but then the hybrid of limonite and epinephelus lanceolatus exhibited about 155% growth at 40 days and 187% growth at 70 days, compared to the purebred limonite (fig. 3).
Example 5 discrimination of hybrid species Using molecular markers
Cutting part of tail fin of Epinephelus coioides, Epinephelus lanceolatus and hybrid thereof, and extracting genomic deoxyribonucleic acid (genomic DNA) by phenol extraction (phenol extraction) method.
The hybrid species were identified by Random Amplified Polymorphic deoxyribonucleic acid (RAPD) technique.
In the random amplification polymorphic deoxyribonucleic acid polymerase chain reaction, 10-mer random primers (Operon Technologies, inc., Alameda, CA.) were used, and 1 genomic deoxyribonucleic acid was used as an object, and 2 primers exhibiting effective band patterns were selected (table 1).
Then, 3 epinephelus lanceolatus, 2 hybrids of the epinephelus lanceolatus and 3 epinephelus lanceolatus are taken as objects, and the corresponding 2 primers are used for verification.
The polymerase chain reaction was performed as follows: after 30ng of genomic deoxyribonucleic acid and 25 μm of random primers were put into 20ul of AccuPower polymerase chain reaction Premix Kit (PCR Premix Kit) (Bioneer, Korea) with a volume, initial thermal denaturation reaction was carried out at 94 ℃ for 5 minutes, and then cycling reaction was carried out 35 times at 94 ℃ for 1 minute, at 36 ℃ for 1 minute, and at 72 ℃ for 2 minutes. Finally, an extension reaction was carried out at a temperature of 72 ℃ for 10 minutes.
The success of the polymerase chain reaction was confirmed by electrophoresis on a 1.5% agarose (agarose) gel stained with GelRed (Biotium inc., USA).
As a result, in the primer (OPA7) of the sequence 1, a specific band of Epinephelus lanceolatus was represented in 320 to 340bp (about 330bp) and 390 to 410bp (about 400bp), and a specific band of Epinephelus fuscoguttatus was represented in 310 to 330bp (about 320bp) and 440 to 460bp (about 450 bp).
The specific bands involved were shared among 2 hybrids of Epinephelus fuscoguttatus and Epinephelus lanceolatus (FIG. 4).
In the primer (OPA8) of the sequence 2, a specific band of Epinephelus lanceolatus is shown in 370-390 bp (about 380bp), a specific band of Epinephelus fuscus is shown in 400-420 bp (about 410bp), and 2 specific bands are shared by 2 hybrids of Epinephelus lanceolatus and Epinephelus lanceolatus (FIG. 5).
Therefore, the primers of the sequence 1 and the sequence 2 can be used for clearly distinguishing the Epinephelus fuscoguttatus and the Epinephelus lanceolatus and hybrids between the Epinephelus fuscoguttatus and the Epinephelus lanceolatus.
TABLE 1
| Primer name | Base sequence (5 '→ 3') |
| OPA7 | GAAACGGGTG |
| OPA8 | GTGACGTAGG |
Sequence listing
<110> Korea institute of ocean science and technology
SOONCHUNHYANG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION
SOONCHUNHYANG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION
Yi le jin
<120> hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and production method thereof
<130>
<150>KR 10-2015-0185564
<151>2015-12-24
<160>2
<210>1
<211>10
<212>DNA
<213> Epinephelus (Epinephelus)
<400>1
<210>2
<211>10
<212>DNA
<213> Epinephelus (Epinephelus)
<400>2
Claims (2)
1. A method for confirming a hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus is characterized by comprising the following steps:
preparing mature eggs of female Epinephelus fuscoguttatus, and after preparing semen of male Epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of male Epinephelus fuscoguttatus to produce fertilized eggs;
a step of incubating and raising the fertilized eggs to produce larval fish;
extracting the produced deoxyribonucleic acid of the larval fish;
mixing the extracted deoxyribonucleic acid of the larval fish with a primer with a sequence 1 or a primer with a sequence 2, and amplifying by utilizing a polymerase chain reaction; and
a step of confirming the product obtained by the above-mentioned amplification by electrophoresis,
the temperature of the hatching tank in the step of producing the fry is 23-27 ℃,
in the step of confirming by electrophoresis, when the primer of sequence 1 is used, the hybrid is judged if bands of 320bp, 330bp, 400bp and 450bp are detected, and in the step of confirming by electrophoresis, when the primer of sequence 2 is used, the hybrid is judged if bands of 380bp and 410bp are detected.
2. A method for confirming a hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus is characterized by comprising the following steps:
preparing mature eggs of female epinephelus lanceolatus, and after preparing semen of male epinephelus fuscoguttatus, performing artificial fertilization between the mature eggs and the semen of the male epinephelus lanceolatus to produce fertilized eggs;
a step of incubating and raising the fertilized eggs to produce larval fish;
extracting the produced deoxyribonucleic acid of the larval fish;
mixing the extracted deoxyribonucleic acid of the larval fish with a primer with a sequence 1 or a primer with a sequence 2, and amplifying by utilizing a polymerase chain reaction; and
a step of confirming the product obtained by the above-mentioned amplification by electrophoresis,
the temperature of the hatching tank in the step of producing the fry is 23-27 ℃,
in the step of confirming by electrophoresis, when the primer of sequence 1 is used, the hybrid is judged if bands of 320bp, 330bp, 400bp and 450bp are detected, and in the step of confirming by electrophoresis, when the primer of sequence 2 is used, the hybrid is judged if bands of 380bp and 410bp are detected.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150185564A KR101829052B1 (en) | 2015-12-24 | 2015-12-24 | A hybrid between longtooth grouper Epinephelus bruneus and giant grouper Epinephelus lanceolatus and a hybridization method thereof |
| KR10-2015-0185564 | 2015-12-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107027661A CN107027661A (en) | 2017-08-11 |
| CN107027661B true CN107027661B (en) | 2020-06-12 |
Family
ID=59356925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611196783.0A Active CN107027661B (en) | 2015-12-24 | 2016-12-22 | Hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and production method thereof |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101829052B1 (en) |
| CN (1) | CN107027661B (en) |
| TW (1) | TWI629362B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102038705B1 (en) * | 2016-12-30 | 2019-10-30 | 순천향대학교 산학협력단 | A hatchability improvement method of hybrid eggs of brown marbled grouper and giant grouper |
| KR101885049B1 (en) | 2018-03-16 | 2018-08-29 | 전상운 | Hybrid production mathod of fishing carp and goldfish |
| KR101886641B1 (en) * | 2018-03-20 | 2018-08-08 | 노기중 | Method for Producing Hybridized Species of Crucian Carp and Carp |
| CN108739537B (en) * | 2018-06-06 | 2021-02-09 | 江苏省淡水水产研究所 | Family breeding method for pink-white channel catfish |
| CN109287533B (en) * | 2018-09-17 | 2024-02-27 | 大连海洋大学 | Large-scale breeding method and equipment for hybrid scallop offspring seeds |
| CN110150188B (en) * | 2019-06-27 | 2021-11-16 | 海南晨海水产有限公司 | Artificial breeding method for hybrid of Epinephelus fuscoguttatus and Epinephelus hybridus |
| CN110521641B (en) * | 2019-10-15 | 2021-02-12 | 中国水产科学研究院黄海水产研究所 | Artificial crossbreeding method for fast-growing grouper |
| CN112655599A (en) * | 2019-11-01 | 2021-04-16 | 海南青利水产繁殖有限公司 | Method for cultivating epinephelus lanceolatus fries |
| CN111149738B (en) * | 2020-02-27 | 2022-01-14 | 海南晨海水产有限公司 | Efficient outdoor ecological pond artificial breeding method for hybrid seeds of Epinephelus coioides and Epinephelus hybridus |
| KR20220116615A (en) | 2021-02-15 | 2022-08-23 | 이일영 | Hybrids of starry flounder and stone flounder and production method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940175A (en) * | 2010-08-09 | 2011-01-12 | 广东省大亚湾水产试验中心 | Method for breeding 'blue spot' hybrids |
| CN105123580A (en) * | 2015-09-06 | 2015-12-09 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for saladfish and lanceolatus |
-
2015
- 2015-12-24 KR KR1020150185564A patent/KR101829052B1/en active IP Right Grant
-
2016
- 2016-12-22 TW TW105142671A patent/TWI629362B/en active
- 2016-12-22 CN CN201611196783.0A patent/CN107027661B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940175A (en) * | 2010-08-09 | 2011-01-12 | 广东省大亚湾水产试验中心 | Method for breeding 'blue spot' hybrids |
| CN105123580A (en) * | 2015-09-06 | 2015-12-09 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for saladfish and lanceolatus |
Non-Patent Citations (2)
| Title |
|---|
| 4种石斑鱼的分子遗传多样性和亲缘关系的RAPD分析;尹绍武等;《海洋学报》;20061130;第28卷(第6期);第119-126页 * |
| Hybridization of Epinephelus species;W.Y.Tseng et al;《Aquaculture》;19830731;第34卷(第1-2期);第177-182页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201730346A (en) | 2017-09-01 |
| TWI629362B (en) | 2018-07-11 |
| KR20170075956A (en) | 2017-07-04 |
| CN107027661A (en) | 2017-08-11 |
| KR101829052B1 (en) | 2018-02-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107027661B (en) | Hybrid of Epinephelus fuscoguttatus and Epinephelus lanceolatus and production method thereof | |
| CN114107522B (en) | Specific marker of female and male genetic sex of oplegnathus punctatus, identification method and kit | |
| CN108841945B (en) | PCR amplification primer, method and kit for rapidly identifying genetic sex of Chinese softshell turtles | |
| CN102134593B (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN111394445B (en) | Indel marker for sex identification of channa maculata and application thereof | |
| CN111088370B (en) | Sex-specific molecular marker primer for Trachinotus ovatus, identification method and application of sex-specific molecular marker primer | |
| CN102578041A (en) | Method for using Huai pigs to cultivate new pig breed and produce hybrid commercial pigs | |
| CN101921850A (en) | Molecular marker screening method of largemouth black bass parent with high hatchability | |
| CN106868112B (en) | molecular identification method for culter alburnus, triangular bream and filial generation thereof | |
| CN113151361A (en) | Method for cultivating crucian carp strain without muscle intermingled bones | |
| CN103798169A (en) | Method for quickly establishing improved breeding system of YY super-male pelteobagrus fulvidraco | |
| CN114657264B (en) | Clarias fuscus sex-specific molecular marker primer and application thereof | |
| Poompuang et al. | A preliminary genetic map of walking catfish (Clarias macrocephalus) | |
| CN104694660B (en) | The method of egg-shaped pompano family paternity test | |
| KR102168817B1 (en) | Method for selection breeding Cyprinus carpio using microsatellite marker | |
| CN105918184B (en) | A kind of method of selection cross all-male Pelteobagrus fulvidraco improved seeds | |
| CN113637765B (en) | Molecular marker for identifying genetic sex of micropterus salmoides and application thereof | |
| CN102912017B (en) | Method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead | |
| CN110331217A (en) | A kind of microsatellite marker paternity identification primer, method and application suitable for bolti, Oreochromis aureus and its cenospecies | |
| CN109880893A (en) | DNA fragment specific and application for mystus nemurus sex identification | |
| Liu et al. | Estimated reproductive success of brooders and heritability of growth traits for large yellow croaker (Larimichthys crocea) using microsatellites | |
| Kocher et al. | Genome mapping and genomics in fishes and aquatic animals | |
| CN112779258A (en) | Large yellow croaker CRISPR/Cas9 gene editing method | |
| JP5452047B2 (en) | Genetic markers linked to the genetic sex of yellowtails, sex discrimination methods for yellowtails, and primers used for sex discrimination methods | |
| CN114574600B (en) | Male molecular marker of alosa sapidissima, and specific primer pair and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |