TWI629362B - An identification method of a hybrid between longtooth grouper epinephelus bruneus and giant grouper epinephelus lanceolatus - Google Patents

Abstract

The present invention relates to a hybrid of a brown grouper and a grouper and a production method thereof, in particular to a hybrid produced by crossing between two types of grouper, a grouper of the grouper grouper and a grouper and a cross, in the family Anidae The production method of the species relates to the production method of the hybrid between the female brown grouper and the male saddlefish grouper and the hybrid between the male brown grouper and the female saddlefish grouper. The present invention can provide a hybrid with a fast growth rate and an excellent morphology by crossing a marketable brown grouper with a fast-growing and marketable saddle grouper. In the hybrid of the present invention, the initial larvae grow fast, so it can solve the problem of biological bait that becomes a big problem when cultivating anchovy, and can contribute to the activation of the Korean farming industry.

Description

Method for confirming hybrids of brown grouper and grouper

The present invention relates to a hybrid produced by crossbreeding between brown grouper and grouper grouper belonging to the grouper Subfamily in the family Anatidae, and a method for producing the hybrid, which relates to female grouper (longtooth grouper, Epinephelus bruneus) and A method of producing a hybrid between a male grouper (Eantphelus lanceolatus) and a hybrid between a male grouper and a female grouper.

The present invention is a method comprising the steps of ensuring mature eggs from females, extracting semen from males, and performing artificial fertilization between the two, thereby producing fertilized eggs, and breeding fertilized eggs into hatching and seeding steps.

Compared with the brown grouper, the hybrid between the brown grouper and the saddle grouper has the advantage of very fast growth.

The family Serranidae is 529 species in 3 subfamilies. Among them, the main culture target species is the subfamily Epinephelinae, which is 159 species in 15 genera. Most of them live in the subtropical Indo-Pacific region.

Anchovy has attracted attention as a high-priced farmed fish, and the production of farming has increased sharply. In 2011, it produced 94,040 tons, and it has a large marketability of 7.3 times in 10 years.

The brown grouper belonging to the object species of the present invention is used as an aquaculture tool Fish species with high added value are called "Oriental Perch" in Korean dialects in Jeju Island, South Korea. They are sold at a high price, and the Hong Kong market, which is the largest market for anchovy, and the demand in China are in high demand.

As a very large fish that inhabits subtropical regions, saddle grouper is attracting attention as a breeding target species in Taiwan due to its fast growth rate and high price, and is gradually being introduced into China.

As a method of mating between two types with different genotypes and phenotypes, hybridization is a technique for producing new species for growth, disease resistance, meat quality, and environmental adaptability that are economically important traits. .

In particular, interspecies hybridization has a high probability of obtaining heterosis through a combination of required qualities, so various attempts are being made (Korean Published Patent No. 10-1999-0045938, Korean Granted Patent No. 10-0844339, Korean Published Patent No. 10-2012-0083990).

On the other hand, the production of various hybrid species targeting the Anatidae is also actively under way.

Representatively, there are dark grouper (dusky grouper (Epinephelus marginatus)) × white grouper (white grouper (E.aeneus)), Mediterranean grouper (goldblotch grouper (E. costae)) × dark spot grouper, Brown-marble grouper (E. fuscoguttatus) × spotted grouper (E. polyphekadion), orange-spotted grouper (E. coioides) × brown-spotted grouper (E. coioides) × Saddle-striped grouper and red-spotted grouper (E. akaara) × spot-spotted grouper, etc.

In particular, a hybrid produced by artificially fertilizing the mature eggs of female brown-spotted grouper and the sperm of a grouper of the grouper is responsible for mass production due to its rapid growth and disease resistance, and Officially registered in the Hong Kong market for sales.

However, so far, the hybrids of Anacidae fishes are only hybrids between subtropical fish species, and the hybridization between Anacidae fishes that live in Korea and subtropical Anacidae fishes has not been achieved.

At the same time, when developing a new species that crosses between the marketable brown grouper and the fast-growing and marketable saddle grouper, along with sales in Korea, it can also be carried out in China, which has the largest consumer market Sales.

[Prior Art Literature] [Patent Literature]

Patent Document 0001: Korean Published Patent No. 10-1999-0045938

Patent Document 0002: Korean Patent No. 10-0844339

Patent Document 0003: Korean Published Patent No. 10-2012-0083990

The purpose of the present invention is to develop new varieties through cross-breeding in order to obtain excellent qualities such as marketability and growth rate of brown grouper and grouper.

In addition, an object of the present invention is to provide a method for hybridization of brown grouper and saddle grouper that have a fast growth rate and can contribute to the activation of the breeding industry.

In order to achieve the above-mentioned object, the present invention provides a method for producing a hybrid of brown grouper and saddle grouper. The method for producing a hybrid of brown grouper and saddle grouper includes: preparing mature eggs of female brown grouper and preparing males After the sperm of the grouper from the saddle belt, the artificial fertilization between the two is carried out to produce fertilized eggs; and the fertilized eggs are hatched and fed to produce larvae.

Furthermore, the present invention provides the growth of a hybrid of brown grouper and grouper The production method of the above-mentioned hybrid production method of brown grouper and saddlefish grouper includes: preparing mature eggs of female saddlefish grouper and preparing semen of male brown grouper, then performing artificial fertilization between the two to produce fertilized eggs Steps; and the steps of hatching and feeding the above fertilized eggs to produce larvae.

In addition, the present invention provides a method for confirming the hybrid species of brown grouper and saddle grouper. The method for confirming the hybrid species of brown grouper and saddle grouper includes the steps of extracting the deoxyribonucleic acid (DNA) of the produced larvae ; Mixing the extracted DNA of the above larvae with the primers of sequence 1 or primers of sequence 2 and the amplification by polymerase chain reaction (PCR); and confirming that the above amplification is obtained by electrophoresis Product steps.

An embodiment of the present invention is characterized in that, in the step of confirming by the above electrophoresis, when the primer of sequence 1 is used, if bands of about 320bp, 330bp, 400bp, and 450bp are detected, it is determined It is a hybrid.

An embodiment of the present invention is characterized in that, in the step of confirming by the above-mentioned electrophoresis, when primers of sequence 2 are used, if bands of about 380 bp and 410 bp are detected, it is determined to be a hybrid.

In addition, the present invention provides hybrids, fertilized eggs, and larvae prepared by the above-mentioned method for producing hybrids of brown grouper and grouper.

The present invention can provide a hybrid with a fast growth rate and an excellent morphology by crossing a marketable brown grouper with a fast-growing and marketable saddle grouper.

In the hybrid of the present invention, the initial larvae grow very quickly, and thus can solve the problem of biological feed that becomes a big problem when cultivating anchovy, and can contribute to the activation of the Korean farming industry.

In addition, the present invention can combat Taiwan, which is the most developed country for the production of Anguidae fish seedlings, and improve the national competitiveness of the Korean aquaculture industry, and can generate enormous income by entering the Anguidae fish farming market.

Figure 1 shows the hatching rate and initial survival rate of the hybrids of brown grouper and grouper.

Figure 2 shows the morphological characteristics of brown grouper, grouper and grouper.

Figure 3 shows the difference in the growth rates of brown grouper and hybrids. The trend line uses an exponential trend line, and the R2 value is disclosed.

Fig. 4 is an electrophoresis photograph of polymerase chain reaction amplification products using primers of sequence 1, GG1, GG2 and GG3 are saddle grouper, LG1, LG2 and LG3 are brown grouper, LG × GG1, LG × GG2 are hybrids .

Fig. 5 is an electrophoresis photograph of polymerase chain reaction amplification products using primers of sequence 2, GG1, GG2 and GG3 are saddle grouper, LG1, LG2 and LG3 are brown grouper, LG × GG1, LG × GG2 are hybrids .

Hereinafter, the present invention will be described in detail based on examples and drawings. The terms, embodiments, drawings, etc. used in the present invention are only used to more specifically clarify the present invention, and help those of ordinary skill in the technical field to which the present invention belongs to understand the present invention, and cannot be construed as claiming protection of the invention The scope is limited to this.

If the technical terms and scientific terms used in the present invention have no other definitions, it means the meaning generally understood by those of ordinary skill in the technical field to which the present invention belongs.

The invention relates to a method for producing a hybrid of brown grouper and grouper The method for producing a hybrid of the brown grouper and the saddlefish grouper includes the steps of preparing mature eggs of the female brown grouper and preparing the semen of the male saddlefish grouper, and then performing artificial fertilization between the two to produce fertilized eggs. ; And the steps of hatching and feeding the above fertilized eggs to produce larvae.

In addition, the present invention relates to a method for producing a hybrid of a brown grouper and a grouper, which includes preparing a mature egg of a female grouper and a sperm of a male grouper The steps of artificial fertilization between the two to produce fertilized eggs; and the steps of hatching and feeding the fertilized eggs to produce larvae.

In addition, the present invention relates to a method for confirming the hybrid of brown grouper and saddle grouper. The method for confirming the hybrid of brown grouper and saddle grouper includes the steps of: extracting the DNA of the produced larvae; extracting The step of mixing the DNA of the above larvae with the primer of sequence 1 or the primer of sequence 2 and performing amplification by polymerase chain reaction; and the step of confirming the product obtained by the above amplification by electrophoresis.

In the step of confirming by the above-mentioned electrophoresis, when the primer of sequence 1 is used, if bands of about 320 bp, 330 bp, 400 bp, and 450 bp are detected, it is determined to be a hybrid.

In the step of confirming by the above electrophoresis, when the primer of sequence 2 is used, if bands of about 380 bp and 410 bp are detected, it is determined to be a hybrid.

In addition, the present invention relates to hybrids, fertilized eggs, and larvae prepared by the above-mentioned method for producing hybrids of brown grouper and grouper.

Hereinafter, the present invention will be described in detail through examples. The following embodiments are only for implementing the present invention, and the content of the present invention is not limited to the following embodiments.

[Example 1. Make sure the brood fish]

In the present invention, as a brown grouper, a female brown grouper matured from an individual with a body weight of 1.5 to 5.2 kg that is collected near Jeju, South Korea and is being domesticated in a broodstock breeding tank is used as a saddle grouper, imported from Taiwan And the mature male saddlefish grouper in the individual with a weight of 54.2 ~ 112.0kg being domesticated in the broodstock feeding tank.

[Example 2. Hybridization]

The abdominal compression method was used to extract eggs and semen from mature female brown grouper and male saddle-head grouper, respectively, and hybridization was conducted by artificial insemination using dry method.

First, in order to obtain high-quality mature eggs of mature female brown grouper, after observing the abdomen, the mature state was confirmed while touching with hands.

25 μg / kg of luteinizing hormone releasing hormone (LHRH) (Sigma, USA) and 1000 IU / kg of human chorionic gonadotropin (HCG) were injected into the abdominal cavity of the mature female brown grouper.

From 36 hours after the injection, it was touched by hand at 4 hour intervals and it was confirmed whether the mature egg was mature, and finally, the mature egg was ensured by pressing the abdomen.

The abdomen compression method was used to extract the semen of male grouper.

It is necessary to prevent the guaranteed mature eggs from being exposed for more than 30 minutes under normal temperature conditions, and after mixing the ensured mature eggs and semen in the egg collection container uniformly, let them stand at room temperature for 1 to 5 minutes, and then add sea water to Start sperm.

After that, add seawater and leave it at room temperature for 5 to 20 minutes. After that, distinguish between floating eggs and sinking eggs.

Among them, only the floating eggs are moved to the egg washing net to wash the residual semen, and then the egg washing net is floated in the water tank to drain the seawater for about 10 to 20 hours.

In the end, only the floating fertilized eggs are collected and moved to the hatching tank.

[Example 3. Hatching and feeding]

Hatching and feeding of larvae were carried out in the same way.

The results of the incubation experiment showed that all of the hatching temperature in the incubator up to 22 ~ 31 ℃ was hatched, but the hatching rate was lower than 25 ℃ and 28 ℃ in 22 ℃ and 31 ℃.

The survival rate of yolk absorbed larvae showed a high value in the experimental group of 25 ℃ and 28 ℃, but did not survive in the experimental group of 22 ℃ and 31 ℃ (Figure 1).

The analysis of the malformation rate of hatched larvae shows that at 25 ° C, the malformation rate is 1.5%, while at 28 ° C, the malformation rate is as high as 15.5%, making the temperature of the appropriate hatching tank preferably 23 to 27 ° C, more preferably It is 23.5 ~ 26.5 ℃.

Rotifers were supplied 2 days after hatching ended after hatching.

As the larvae grow, compound feed is supplied with Artemia larvae. Feeding water uses filtered seawater, which is maintained exponentially at the beginning of the feeding period, and the amount of water changed in one day after supplying Artemia is maintained at 30-50%. The excrement in the water tank was removed through the siphon from the step of supplying Artemia.

[Example 4. Growth rate survey]

The size of the larvae after hatching, the size of the larvae after yolk absorption, and the growth rate at 3, 5, 7, 12, 12 and 17 days of hatching were investigated (Figure 2).

The measurement was carried out on the basis of the full length, and in order to compare the growth of the corresponding hybrids, the comparison was made on the basis of the full length of the pure grouper of brown grouper.

There was no significant difference between the pure grouper of brown grouper and the hybrid of brown grouper and saddle grouper until 20 days after incubation, but after that, the hybrid of brown grouper and grouper of saddle grouper was 40 days Showing about 155% growth at 70 The day showed 187% growth (Figure 3).

[Example 5. Discrimination of hybrids using molecular markers]

Taking brown grouper and saddle grouper and the hybrid between the two as the object, a part of the tail fin was cut, and the genomic DNA was extracted by phenol extraction method.

Random amplified polymorphic DNA (RAPD, Random Amplified Polymorphic DNA) technology was used to identify hybrids.

10-mer random primer (Operon Technologies, Inc., Alameda, CA) was used in the random amplified polymorphic DNA polymerase chain reaction .)), Using a total of three genomic DNA as a target, first polymerase chain reaction was performed to select two primers exhibiting effective band patterns (Table 1).

Afterwards, 3 saddle-spotted groupers, 2 brown grouper and hybrids of saddle-striped grouper and 3 brown grouper were used as the targets, and the corresponding 2 primers were used to verify.

The polymerase chain reaction was carried out as follows: 30 ng of genomic deoxyribonucleic acid and 25 μM of random primers were placed in the AccuPower polymerase chain reaction premix kit (PCR Premix Kit) (Bioneer, Korea) ) Company), after the initial thermal denaturation reaction at 94 ° C for 5 minutes, a loop reaction at 94 ° C for 1 minute, 36 ° C for 1 minute, and 72 ° C for 2 minutes 35 times. Finally, the extension reaction was carried out at 72 ° C for 10 minutes.

In a 1.5% agarose gel stained with GelRed (Biotium Inc., USA), electrophoresis was performed to confirm the success of the polymerase chain reaction.

As a result, among the primers of sequence 1 (OPA7), the specific bands of saddle banded grouper appeared in 320 ~ 340bp (about 330bp) and 390 ~ 410bp (about 400bp), and in 310 ~ 330bp (about 320bp) and 440 ~ The 460bp (approximately 450bp) showed a specific band of brown grouper.

The above-mentioned specific bands were shared among two hybrids of brown grouper and grouper grouper (Figure 4).

In the primer of sequence 2 (OPA8), the specific band of saddle-striped grouper was presented at 370 ~ 390bp (approximately 380bp), and the specific band of brown-spotted grouper was presented at 400-420bp (about 410bp). The two hybrids with grouper shared two specific bands (Figure 5).

Therefore, the primers of sequence 1 and sequence 2 can be used to clearly distinguish between brown grouper and saddle grouper and the hybrids between the two.

<110> Yoon Jin-jin, Industry-Academy Cooperation Team, Mokpo University, South Korea Institute of Marine Science and Technology

<120> Hybrids of brown grouper and grouper grouper and production method

<130>

<150> KR 10-2015-0185564

<151> 2015-12-24

<160> 2

<210> 1

<211> 10

<212> DNA

<213> Epinephelus

<400> 1

<210> 2

<211> 10

<212> DNA

<213> Epinephelus

<400> 2

Claims (1)

A method for confirming the hybrid of brown grouper and saddle grouper, which includes: preparing mature eggs of female brown grouper and preparing semen of male saddle grouper, and performing artificial fertilization between the two to produce fertilized eggs Steps of incubating and feeding the fertilized eggs to produce larvae; extracting the DNA of the produced larvae; extracting the DNA of the larvae and the primer of sequence 1 or the primer of sequence 2 The steps of mixing and performing amplification by polymerase chain reaction; and the step of confirming the product obtained by the above amplification by electrophoresis. The temperature of the hatching tank in the step of producing larvae is 23 to 27 ° C. In the step of confirming by the above electrophoresis, when the primers of sequence 1 are used, if the bands of 320 bp, 330 bp, 400 bp, and 450 bp are detected, the hybrid is determined, and when the primers of sequence 2 are used, if the detection The bands up to 380bp and 410bp are determined as hybrids.