KR20160131369A

KR20160131369A - A Method of Mass Production of the Albino Misgurnus anguillicaudatus

Info

Publication number: KR20160131369A
Application number: KR1020150063613A
Authority: KR
Inventors: 방인철; 문신주; 한승희
Original assignee: 순천향대학교 산학협력단
Priority date: 2015-05-07
Filing date: 2015-05-07
Publication date: 2016-11-16
Also published as: KR101726812B1

Abstract

The present invention is to provide a method capable of massively producing albino Misgurnus anguillicaudatus by screening an F1 group having 100% albino trait by breeding natural albino Misgurnus anguillicaudatus broodstork; and screening an F2 group expressing albino trait at 100% by breeding the F1 group produced above to establish a mass production line in which albino expression rate of all generation from natural broodstork to F1 and F2 is fixed at 100%. More particularly, the present invention comprises the following steps of: (a) collecting albino Misgurnus anguillicaudatus or natural broodstork assumed as albino Misgurnus anguillicaudatus; (b) determining a species of the collected natural broodstork; (c) inducing mature of natural broodstork, which is determined as albino Misgurnus anguillicaudatus in the species determination step; (d) producing an F1 group through artificial spawning and breeding of the individual mature-induced natural broodstork; (e) identifying albino trait expression rate in the F1 groups; (f) breeding and inducing mature only the F1 group having albino trait expression rate identified in step (e) of 100% and producing an F2 group by individually breeding the mature-induced F1 broodstork; (g) identifying albino trait expression rate in the F2 groups; (h) establishing a mass production line by screening the F2 group only having the albino trait expression rate identified in step (g) of 100%; and (i) mass producing albino Misgurnus anguillicaudatus by using individuals included in the established mass production line as broodstork.

Description

[0001] The present invention relates to a method for mass production of an albino loach (Misgurnus anguillicaudatus)

The present invention relates to a method for securing an albino trait in the production of albino loofah, thereby enabling mass production.

The ornamental fish are an ever-growing industrial sector with annual added value of several tens to hundreds of times higher than that of edible fish, and it is possible to generate huge profits by developing rare species production techniques.

Recently, the development of ornamental fish using native species in the international environment, which can act as barriers to bio-trade due to the ABS convention, is expected to become a profit model that creates high value-added.

In the market of ornamental fish, albino fish are highly mutated and have a high probability of emergence. They have high scarcity and merchantability and are well known in various fields.

Misgurnus anguillicaudatus is a small benthic fish belonging to the Genus Misgurnus of the subspecies of the sand, and is distributed in the whole of Korea's freshwater, China, and Japan. It is mainly used for the flow of water such as swamps, rice paddies, They live on weak or stagnant clay, where they eat algae and organic matter.

The common loach is very similar to the loach, which is similar to the loach, and the loach is longer and larger than the loach, but the length of the loach is larger or larger than the loach. .

Especially, in the case of albino loach, there is no coloration due to melanin pigment deficiency, and it is impossible to distinguish species by external form. Therefore, it is difficult to classify loach and its external morphological characteristics, and species identification by molecular biology must be performed.

GOLDEN LOACH, currently sold abroad, is an incomplete albino that has black eyes with a golden color and is traded at a relatively high price. Albino Mudfish (red mudfish) with red eyes in the golden color of the domestic albino mudfish has higher world-class competitiveness because of its higher commercial value.

However, the spontaneous Albino Mudfish appearing in Korea appears at a very low frequency in nature, making it difficult to obtain a large number of siblings.

The artificial seedling production method and breeding technique of general loach were secured and supplied, but the genetic characteristics of albino loach of albino loach have not been studied in detail yet.

In addition, the inventors of the present inventors have not produced a large amount of albino mudfish in Korea. As a result of previous researches, even when producing albino fry (females and males) using females, 100% (Genetic phenomenon of albino trait of Misgurnus anguillicaudatus ), Korean Society of Fisheries Science Spring Conference abstract.

As a prior art document related to the production method of the muddy seedling produced in Korea, there is a production method of a mudfish reverse transgenic product (Korean Patent Registration No. 0243027), and prior art documents of a mudfish, which is a similar species, Korean Patent Registration No. 1169013), a primer for identifying the origin of loach and an identification method using the primer (Korean Patent Registration No. 1380779), and a method for producing a 4-labeled loach (Korean Patent Publication No. 2000-0015918). The technology related to the loach, which is a similar species, is mainly performed in Japan, and it is known that a method of culturing the loach, a method of culturing the loach (Japanese Unexamined Patent Application Publication No. 2013-74844), a method of cultivating loach, (Japanese Unexamined Patent Application Publication No. 5-336860), methods for producing loach seedlings (Japanese Unexamined Patent Application Publication No. 8-131014), loach culture methods (Japanese Unexamined Patent Application Publication No. 2006-340656), loach loach culture methods -116887) and a method for identifying an individual using the loach Cytb gene (Japanese Patent Laid-Open Publication No. 11-266869).

In order to solve the above problems, the present invention provides a method for screening F1 group having 100% albino phenotype by crossing between wild-born albino mulberry siblings and F2 group To provide a method for mass production of albino mudfish by constructing a mass production line in which the expression rate of albino is fixed at 100% in all generations from wild-born siblings to F1 and F2.

In order to accomplish the above object, the present invention provides a method for mass production of albino loofah, comprising the following steps.

(a) collecting a wild-born parent estimated to be albino loofah or albino loach

(b) a step of discriminating the species of the collected wild-

(c) inducing maturation of the wild-born parent identified as albino loach in the step of discriminating the species

(d) artificially scattering and modifying the maturation-induced wild-born parent by individual to produce F1 groups

(e) confirming the expression rate of the albino gene in the F1 groups

(f) breeding and maturing only the F1 group having the expression rate of 100% of the albino gene identified in the step (e), and mating each maturation-induced F1 parent individual to produce F2 groups

(g) confirming the expression rate of the albino gene in the F2 groups

(h) constructing a mass production line by selecting only the F2 group having an expression rate of 100% of the albino gene identified in the step (g)

(i) mass production of albino loofah using the individuals included in the constructed mass production line as a parent

The present invention has the effect of stably mass-producing a complete albino loofah with red eyes and a golden body color by 100% expression and fixing of the albino trait to the next generation in the albino loofah with high commercial value.

Figure 1 shows the result of species identification using the RAG 1 region of albino loach and albino loach
Fig. 2 shows the results of species identification using the Cyto B region of albino loach and albino loach
Fig. 3 is a graph showing the relationship between the VIE tag
Fig. 4 shows the results of the presence or absence of expression of an albino gene on the 2nd day after F1 hatching (left: common loofah, right: albino loofah)
FIG. 5 shows the frequency of expression of the albino transcript (50% left, 100% right albino expression)
FIG. 6 is a graph showing the results of an albino loofah F2
Fig. 7 is a diagram illustrating the construction of a mass production line of the albino loach
Fig. 8 is a schematic diagram of a mass production method of albino loach

The present invention relates to a method for classifying albino lozenges and albino lozenges collected and secured in nature using a taxonomic and genetic classification method and then mass-producing the albino lozenges by fixing the albino traits at a later stage using the identified albino loofahs As a method, 100%, 50%, and 25% of the albino traits were expressed in F1 according to the genotype and phenotype of the siblings, and the F1 group in which the albino trait was expressed in 100% was selected and the albino trait was 100% To produce an F2 that is fixed at a high level, thereby producing a stable mass production of albino loach.

Hereinafter, the present invention will be described in detail for each step.

(a) collecting a wild-born parent estimated to be albino loofah or albino loach

This step is collecting complete albino individuals with red eyes and a golden body color. Because albino loach or albino loach is difficult to distinguish species from the naked eye once, it collects all the objects that are presumed to be albino loach or albino loach. The reason for collecting only complete albino individuals with red eyes and golden color is as shown in Table 1 below (preliminary experiment results of albino production rate by crossing group according to eye color) This is because the incomplete albino mudfish that has been mated with the complete red alpine mudfish produces only common loquat seedlings in later generations.

Mating experiment group (female x male) Analysis Mari Albino offspring Generic offspring Albino ratio (%) Red eyed pecker ×
Red eye muddy Experiment 1 100 100 0 100 Experiment 2 100 48 52 48.0 Red eyed pecker ×
Black eye muddy Experiment group 3 150 0 150 0 Experiment group 4 30 0 30 0 Red eyed pecker ×
General loofah Experiment group 5 200 0 200 0 Experiment group 6 23 0 23 0

(b) a step of discriminating the species of the wild-born parent

This step is a step to prevent the artificial hybrid induction by the loach by distinguishing clearly the species of the alopecia or the loach among the collected wild relatives and selecting the loach.

There are morphological and molecular biologic methods for classifying loach and loach. First, the morphological classification method is that the loach is rounder than the loach, the length of the loach is short, and regular small dots are arranged on the caudal fin and dorsal fin, while the loach is flat on the body and long on the base of caudal fin The point is unclear.

However, in the case of albino, it was impossible to distinguish the body color due to absence of melanin pigment due to albinism (albinism), and in case of metrical traits, The morphological analysis of mudfish and albino loach, as shown in the morphological analysis results, is different only for the length of the two heads, and there is no difference in the other traits.

Analytical trait Albino Mudfish (n = 17) Albino loach (n = 6) ANOVA
( p < 0.05 ) BD / SL 12.82 ± 2.97 12.25 + 1.84 0.68 BW / SL 7.92 ± 1.53 6.06 ± 1.83 0.31 CPD / SL 12.97 ± 3.25 11.09 + - 2.48 0.23 CPL / SL 32.72 + - 8.13 32.24 + - 6.93 0.90 PL / SL 21.27 ± 3.63 17.56 ± 2.14 0.35 HL / SL 15.94 + 2.81 15.56 ± 0.73 0.76 ED / HL 19.21 + - 3.36 17.27 ± 2.11 0.22 EW / HL 38.24 + - 6.63 33.63 + - 4.70 0.15 DPP / SL 41.09 + - 3.15 41.91 + 1.42 0.56 CPD / CPL 40.32 + - 6.71 34.94 + - 5.42 0.11 LB / HL 43.80 7.78 51.77 ± 8.96 0.06

PL / SL: Pectoral fin origin / body length, HL / SL: 2/3/4/2/4 / / Length, ED / HL: glasses / two,

EW / HL: Binocular interval / two, DPP / SL: Dorsal fin origin / length from pectoral fin,

CPD / CPL: US Army / Unknown disease, LB / HL: Receipt length / two

Next, the molecular biology classification method is to analyze the nucleotide sequence of the RAG 1 region of Cyto B or nucleotide DNA of mitochondrial DNA. Specifically, the obtained DNA of the wild-born albino isolate was subjected to PCR using the primers shown in Table 3 (information on the primers used for identification of albino loach and albino lozenges), followed by analysis of the base sequence, It is a method to distinguish the registered loach and loach by comparison with Cyto B or RAG 1 homologous region sequence by neighborhood binding analysis. As shown in FIG. 1 and FIG. 2, the species classification of the mudfish and loach was clarified by nucleotide sequence analysis of the Cyto B or RAG 1 region, and the albino mudfish and albino lozenges were identified by molecular biology It is possible to classify the species accurately.

Primer name 5 'sequence 3' Annealing temp (℃) COB Forward 14513 TGR CTT GAA RAA CCA YCG TTG T 55 Reverse 15868 GTG GGA GTT ARA ATC TC 55 RAG I Forward 1492 AGY CAR TAY CAY AAR ATG TA 54 Reverse 2476 TCC TAG AAG ATY TTG TAR AA 54

This step is a step to feed the mixed feed and the raw material (worm worm, persimmon) at the water temperature of 20 ℃ and to control and maturity of the wild unidentified siblings. The feed was circulated and filtered at 10 times / day for 1 hour in a dark circulation type water tank (1,000 × 60 × 40 cm) treated with ammunition. Feeds were fed at intervals of about 2 hours in the order of compound feed - yarn worm - After 1 hour of feeding, feeds that have not been consumed are collected in the breeding tank and half of the breeding water is returned.

The induction of maturation is carried out by injecting LHRH-a at a dose of 50 μg per kg of the infant, selecting and harvesting the abdomen slightly, and raising the temperature of the raising water by 3 ° C. and matured for 30 days at 23 ° C. During maturation induction period, 2 g of vitamin E and C were fed to the compound feed by the adsorption of 2 g per kg of feed, and 1 g of vitamin C and E were dissolved in 200 ml of water per 10 g of each earthworm and perch, And induce maturation by feeding.

(d) step of producing F1 groups by artificially scattering and modifying adult-induced wild-born individuals

This step is a step of artificially scattering albino mulched alien matured individuals matured in step (c). First, the VIE tag as shown in FIG. 3 was performed for each female and male individual so as to distinguish the individuals. Then, the mature hormone Ovaprim and the egg yolk inducing agent, HCG, were injected at 0.5 ml / kg and 10,000 IU / After 12 hours, the F1 group is produced by bulking and culling by abdominal compression method and modifying by dry method.

(e) confirming the expression rate of the albino gene in F1 groups

This step is to confirm the expression rate of the albino expression of F1 groups produced by modification in step (d). The reason for confirming the expression rate is that, even if only the siblings having complete albino traits are crossed with each other, since the offspring whose albinogen traits are not expressed at 100% can be produced according to the genotype of the siblings, This is to select households that can be 100% expressed. As an example for this purpose, it is possible to observe the development process immediately after hatching of the F1 groups to quickly detect the expression of the albino trait.

The development process of egg albumen and hatching of Albino Mudfish occurs at the same stage as that of conventional mudfish. On the first day after hatching, black vesicles appear on eyes and body side, but Albino Mudfish does not show black vesicles On the second day after hatching, as shown in FIG. 4, the eyes are colored in black, whereas the albino loquat is not colored. Therefore, the expression rate of the albino trait during egg development can be confirmed based on this .

(f) inducing only the F1 group having an expression rate of 100% of the albino gene to induce sarcoma and maturation, and mating the maturation-induced F1 parent individually to produce F2 groups

This step is a step of breeding and maturing only the F1 group having the expression rate of 100% of the albino transcripts identified in step (e), and then producing F2 using this as a parent. Seed breeding is provided by sea rotifers (rotifers) for F1 size less than 1 cm at 20 ° C to 25 ° C water temperature, and sea tangle rotifers and alfalfa larvae for 1 to 3 cm. Feed the feed, and feed the compound feed after 5 cm. The food supply is supplied three times a day, and the water supply is three times / day or more.

When the size of the individual F1 groups grows to about 15 cm or more, F1 is matured in the same manner as that of the wild-born in step (c), and F1 matrices induced by artificial scattering And modify them to produce F2 groups on an individual basis.

(g) confirming the expression rate of the albino gene in F2 groups

This step is a step for confirming the expression rate of the albino gene in the F2 groups produced by each parent by modifying in step (f). For this, the expression rate of the albino gene during egg development can be confirmed in the same manner as in the step (e).

(h) Step of selecting a F2 group having an expression rate of 100% of the albino gene to construct a mass production line

This step is the step of constructing a mass production line that can produce the next generation with the albinotype being fixed at 100% expression rate. For this purpose, a F2 group with an expression rate of 100% of the albino expression is selected, and a mass production line can be constructed by sequentially selecting F1 individuals producing such F2 group and wild birds producing such F1 individuals. With the mass production line constructed above, it is possible to mass produce the albino loofah with a 100% probability of expressing the albino trait.

(i) Mass production of albino loofah using the individuals included in the established mass production line as a parent

This step is a step of mass-producing alopecia having an albino character using the albino loquat individuals contained in the mass production line constructed in step (h) as a parent. Specifically, it is possible to continuously produce offspring having 100% albino traits fixed using wild-born siblings or F1 individuals or F2 individuals included in the established mass production line, or to reproduce and progeny these offspring Which can mass produce 100% albino loofah.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the present invention.

(Example 1)

The wild-type Ovaprim and the egg-laying inducer, HCG, were given 0.5 ml / kg and 20,000 IU, respectively, to the adult male and 2 male males, / kg, and after 12 hours, they were collected and collected by abdominal compression method, and modified by the dry method to produce a total of 6 groups of F1.

(Example 2)

As a result of examining the expression rate of the albino trait to the total of 6 groups of F1 produced, the F1 group in which the albino trait was expressed in 100% was the group of the wild type female of VIE tag and the wild male of VIE tag 1 , F1 groups produced in the experimental group that crossed the VIE tag no. 2 wild female and the VIE tag no. 2 wild male. The albino ratio was 45.0% and 49.0%, respectively, and the wild-type female VIE tag 3 and wild male VIE tag 1 or 2 were obtained. Albino ratio was 48.0% and 48.5% in the crosses.

All of these strains were only able to produce offspring with 100% albino traits, even if they were mated only to wild-born siblings with complete albino traits. In addition, the expression rate of the F1 albino gene was different according to the wild female used, even if the wild male was the same. VIE tag # 2 produced 100% F1 albino, while VIE tag # 1 and # 3 produced only about 50% of albino F1 (Table 4) 5]).

Wild-born mating group Produced F1 Albino
Expression rate (%) female cock analysis
Marie Albino
individual Normal
individual VIE tag 1 VIE tag 1 60 27 33 45.0 (F1 group 1) VIE tag 2 200 98 102 49.0 (F1 group 2) VIE tag 2 VIE tag 1 150 150 0 100 (F1 group 3) VIE tag 2 200 200 0 100 (F1 group 4) VIE tag 3 VIE tag 1 100 48 52 48.0 (F1 group 5) VIE tag 2 130 63 67 48.5 (F1 group 6)

(Example 3)

The siblings of two F1 groups (Group 3, Group 4), in which the albino trait was expressed as 100% in Example 2, were matured and matured-derived siblings were seeded in Table 5 (results of crossing of Albino Mulberry F1 parent- To produce F2, and the expression rate of the albino gene in the produced F2 groups was confirmed. Mating the females of group 3 with the males of group 3, the females of group 3 and the males of group 4 resulted in 100% of the albino-derived F2, and the females of group 4 and group 3, the females of group 4 As a result of crossing the male of group 4, the albino-type F2 was also produced in 100%, and the two F1 groups (group 3, group 4) in which the albino character was expressed in 100% (See [Table 5] and [Figure 6]). Therefore, the family from the wild female VIE tag 2 and the wild male VIE tag 1 to 2, the F1 group 3 to 4 produced from these wild-born siblings, and the F2 group 1 to 4 produced from these F1 groups 3 to 4, And can be selected as a mass production line capable of mass production (see FIG. 7).

F1 breeding group F2 produced Albino
Expression rate (%) female cock analysis
Marie Albino
individual Normal
individual Group 3 Group 3 120 120 0 100 (F2 group 1) Group 4 100 100 0 100 (F2 group 2) Group 4 Group 3 64 64 0 100 (F2 group 3) Group 4 150 150 0 100 (F2 group 4)

(Example 4)

To re-verify the genotype of the siblings for producing albino loofah F2, male of two F1 groups (Group 3, Group 4) used in Example 3 and 3 wild-females used in Example 2 (VIE tag 1-3 ), And reverse transgenic offspring were produced, and the expression rates of the albino transgenic lines of the reverse transgenic offspring were confirmed.

As a result of reverse mating of 3 male and 3 female of group 3 and 4 in F1 crossbreeding group, the expression rate of albino expression was 45% and 49% in wild type female 1 (VIE tag 1) (VIE tag 2), the expression rate of albino transcripts was 100%. In the reverse transcription of wild-type female 3 (VIE tag 3), the expression rate of albino transcripts was 46.7% and 45.0% (See [Table 6] (results of reciprocal cross-breeding of wild-born female and F1 male with the albino trait)).

Based on the experimental results, the wild female VIE tags 1 and 3 are suitable for fixing the albino gene, which shows only about 50% of the albino dielectric constant to F1, even though the outer appearance has the perfect albino loach phenotype with red eyes and golden body color , And it was finally verified that the wild female VIE tag 2 is a suitable strain for the albino characterization which can produce 100% albino offspring having a complete albino loach phenotype with red eyes and golden color .

Reverse mating group F2 produced Albino
Expression rate (%) Wild female F1 male analysis
Marie Albino
individual Normal
individual VIE tag 1 Group 3 60 33 17 45.0 Group 4 200 102 17 49.0 VIE tag 2 Group 3 60 60 0 100 Group 4 20 20 0 100 VIE tag 3 Group 3 30 14 0 46.7 Group 4 40 13 0 45.0

Claims

(a) collecting a wild-born parent estimated to be albino loofah or albino loach;
(b) determining the species of the collected wild-born parent;
(c) inducing maturation of the wild-born parent identified as albino loach in the step of discriminating the species;
(d) artificially scattering and modifying the maturation-induced wild-born parent by individual to produce F1 groups;
(e) confirming the expression rate of the albino gene in the F1 groups;
(f) breeding and maturing only the F1 group having the expression rate of 100% of the albino gene identified in the step (e), and mating each maturation-induced F1 parent individual to produce F2 groups;
(g) confirming the expression rate of the albino gene in the F2 groups;
(h) constructing a mass production line by selecting only the F2 group having an expression rate of 100% of the albino gene identified in the step (g);
(i) mass-producing the albino loach using the individuals included in the mass-produced line as a parent.

The method according to claim 1,
Wherein the step (b) is a molecular biologic method.

The method according to claim 1,
The induction of maturation in the step (c) or (f) is carried out by selecting and individually collecting the abdominal parts and then injecting LHRH-a at 50 μg / kg of the infected human body and incubating at 23 ° C. for 30 days. And 2 g of vitamin E and C per 10 g of the compounded feed are supplied by adsorption, and 1 g of vitamins C and E per 10 g of earthworm and persimmon are dissolved in 200 ml of water, followed by adsorption for 4 minutes to induce maturation. Mass production method of Mudfish.

The method according to claim 1,
Wherein the expression rate of the albino gene in step (e) or (g) is determined by whether or not black vesicles are produced in the first day after hatching.

The method according to claim 1,
Further comprising, after the step (g), a step of crossing the wild-born parent of step (d) and the F1 parent of step (f) to verify the genotype of the wild-born parent.

An albino loofah produced by the mass production method of any one of claims 1 to 5.