KR101135443B1 - Breeding method for genetically improved olive flounder which grows faster - Google Patents

Abstract

The present invention relates to a method of breeding flounder, more specifically, to constitute a mother population having genetic diversity, from which artificial insemination by artificial mating guidelines based on genetic selection and genetic flexibility relationship Through a method for breeding the flounder. Since the breeding flounder produced using the breeding method of the present invention has been confirmed that the growth is faster and improved body shape compared to the conventional farmed and natural flounder, the flounder breeding method of the present invention reduces the production cost of the flounder culture and commercialization of the flounder It will be useful for improvement.

Flounder, starter breeding, body shape improvement, growth efficiency increase

Description

Breeding method for genetically improved olive flounder which grows faster}

The present invention relates to a method of breeding flounder.

Few studies have been reported on the improvement of fish breeds using selective breeding such as 10% growth rate per generation in carp, rainbow trout, Atlantic salmon and tilapia. In the case of Japan, in 1964, the traditional selection breeding method of red snapper was shortened from 36 months before the selection to 19 months for the fifth generation. Did not lead to. Full-scale industrial research under the breeding program has been carried out in Norway in 1971, since the breeding research through the continuous breeding through the salmon breeding program has been carried out in Norway, and the breeding program based on the maintenance of genetic diversity by combining biotechnology techniques with breeding. The quality seedlings, which have been developed and increased by 12% per household, are supplied and account for more than 70% of the salmon produced in Norway. In addition, tilapia has resulted in an increase of 10-15% per generation through traditional selection breeding, and recently developed varieties with an increase of 20-30% per generation through breeding programs based on maintaining genetic diversity. A study is underway.

The breeding technology of halibut farming in Korea is the world's best, but there has been no research on breeding for the development of competitive high-quality breeds compared to the rapid development of breeding technology for the past 20 years since the aquaculture industry started. In particular, it is possible to select only mothers with good external growth, not based on accurate genetic evaluation, or random mating in a limited group of mothers, such as the number of mothers contributing to the next generation, imbalance of parental ratio, household size, etc. Influence of factors is known to typically reduce genetic diversity across culture populations (Sekino et. al ., 2003, Aquaculture , 221: 255-263; Kang et al ., 2006, Aquacul. Res ., 37: 701-707). Reduction of genetic diversity in aquaculture populations is not only due to reduced egg production, lower survival rates, and lower growth rates (Allendorf and Ryman, 1987, Genetic management of hatchery stocks. (In) N. Ryman and F. Utter (ed), Population Genetics and Fishery) Management. University of Washington Press, USA, 141-159) such that weaken the disease resistance and reduce the adaptability to the environment (Carvalho and Haeser, 1994, Rev . Fish Biol . Fish ., 4: 326-350), is a major cause of declining aquaculture productivity. Therefore, for sustainable breeding, it is very important to secure the genetic diversity of the first mother population and to maintain the genetic diversity of the mother population so that it can be maintained through repeated generations. In addition, continuous and integrated breeding programs are needed, such as mating combinations of mother candidates and household production, based on accurate genetic assessment of individual and household breeding traits.

The development of new varieties by selection breeding, where the desired traits are gradually improved over time, will not only take long periods of time, but also the maintenance and maintenance of a certain number of mother populations, the production and breeding of offspring, and the exact inheritance of them. It is very rare that industrial performance is caused by difficulties such as capacity assessment. In particular, existing farming sites select only the leading group with apparent growth without eye measurement, and the mother populations selected without regard for genetic diversity are generated by uncontrolled random breeding in the tank. Due to the limited number of spawning mothers and the 1: 1 imbalance and inbreeding due to multiple breeding, the possibility of genetic deterioration is serious and the productivity of aquaculture is decreasing recently.

Conventionally, the present inventors have disclosed the contents of a method for analyzing genotype of each individual of the flounder in Korean Patent Publication No. 10-2009-0069898. Accordingly, by applying the genotyping method using the composition of the patent it will be possible to select breeding considering the genetic characteristics of the flounder, such as genetic analysis, individual identification and paternity of the flounder population.

The present invention solves problems such as growth retardation, malformation, and culling caused by individual growth differences, which have a great influence on the flounder culture productivity, and quickly and largely new breed of flounder with excellent body shape to enable breakthrough productivity improvement for securing flounder culture competitiveness. We wanted to develop. Therefore, while maintaining the diversity of breeding flounder populations through individual analysis and family management using genetic analysis and paternity technology, the mother selection and the planned mating guidelines based on heredity evaluation are designed based on accurate measurement data. The present invention has been completed by producing improved breeding flounder of growth and body shape through artificial insemination between excellent mothers.

It is an object of the present invention to provide a breeding method for enhancing the growth efficiency of the flounder.

Still another object of the present invention is to provide a breeding flounder with improved growth efficiency.

In order to achieve the above object,

1) analyzing genotypes of the flounder female population and the male population, respectively;

2) calculating genetic flexibility between the individual of the female group and the male group according to Equation 1 below;

&Quot; (1) "

: Genetic relationship between x and y entities

P: frequency of the i allele of subject x

Px: 1 if the i allele of x is homozygous, 0.5 if heterozygous (i = even if i = even, 1 if equal to i-1, 0.5 if different; i + 1 if i = odd 1 if equal, 0.5 if different)

Py: The i allele of x is 0 compared to the two alleles of y and 0 if there is no equal, 0.5 if there is one, and 1 if there are two equals (i = i-allele of x and i- of y Compare 1 and i alleles; if i = odd, compare i alleles of x and i and i + 1 alleles of y)

3) selecting a male individual from the female population in the female population and a male population in which the calculated genetic relationship is 0.2 or less;

4) selecting the first to third excellent individuals having the best expression traits among the male individuals selected in step 3);

5) mating the male individual selected in step 4) and the female individual of step 3) through artificial insemination;

6) breeding the F1 generation produced by the crossing of step 5) for 150 to 200 days;

7) confirming paternity of the F1 generation;

8) measuring the breeding value for each trait of the F1 individuals identified in the paternity of step 7);

9) selecting female and male individuals having higher rankings among the households produced as a result of the crossing of step 5), and then inbringing them; And,

10) It provides a breeding method for increasing the growth efficiency of the flounder comprising the step of assaying the growth efficiency of the breeding F2 flounder produced by the cross of step 9).

The present invention also provides a breeding flounder improved growth efficiency breeding by the above method.

Since the breeding flounder produced using the breeding method of the present invention has been confirmed that the growth is faster and improved body shape compared to the conventional farmed and natural flounder, the flounder breeding method of the present invention reduces the production cost of the flounder culture and commercialization of the flounder It will be useful for improvement. The fast growing breeding flounder provided by the present invention not only improves feed efficiency but also reduces the cost of farming such as labor costs, chemical costs, power costs, oil costs, consumables, and depreciation costs due to the shortened growth period. The ripple effect is likely to be quite high.

Hereinafter, the present invention will be described in detail.

Hereinafter, the term of this specification is demonstrated.

Selective sarcoma is a breeding program that takes advantage of a phenotypic variation inherited from a parent by selecting and breeding a particular or superior one. The present inventors have tried to breed halibut with fast growth rate and improved body shape by selecting halibut with excellent expression traits from a population of mothers with various genotypes.

The term "genetic parameter" refers to the numerical value obtained by actual measurement in a sample when interpreting genetic quantitative traits, It refers to the genetic properties of the population.

In addition, the term "relationship coefficient" is an index indicating the similarity of the genetic makeup between two individuals. Used to indicate the degree of kinship between individuals in the breed or lineage of livestock or experimental animals.

In addition, the term “breeding value” refers to the mean value of a particular quantitative trait for a particular hybrid subscale.

In addition, the term “heritability” refers to the variance of variances expressed by quantitative traits as variances, divided into genetic and environmental variances, and the degree of genetic variance for phenotypic variance is called heritability. do.

The present invention

1) analyzing genotypes of the flounder female population and the male population, respectively;

2) calculating genetic flexibility between the individual of the female group and the male group according to Equation 1 below;

&Quot; (1) "

: Genetic relationship between x and y entities

P: frequency of the i allele of subject x

Px: 1 if the i allele of x is homozygous, 0.5 if heterozygous (i = even if i = even, 1 if equal to i-1, 0.5 if different; i + 1 if i = odd 1 if equal, 0.5 if different)

Py: The i allele of x is 0 compared to the two alleles of y and 0 if there is no equal, 0.5 if there is one, and 1 if there are two equals (i = i-allele of x and i- of y Compare 1 and i alleles; if i = odd, compare i alleles of x and i and i + 1 alleles of y)

3) selecting a male individual from the female population in the female population and a male population in which the calculated genetic relationship is 0.2 or less;

4) selecting the first to third excellent individuals having the best expression traits among the male individuals selected in step 3);

5) mating the male individual selected in step 4) and the female individual of step 3) through artificial insemination;

6) breeding the F1 generation produced by the crossing of step 5) for 150 to 200 days;

7) confirming paternity of the F1 generation;

8) measuring the breeding value for each trait of the F1 individuals identified in the paternity of step 7);

9) selecting female and male individuals having higher rankings among the households produced as a result of the crossing of step 5), and then inbringing them; And,

10) It provides a breeding method for increasing the growth efficiency of the flounder comprising the step of assaying the growth efficiency of the breeding F2 flounder produced by the cross of step 9).

First, the present inventors collected wild flounder as well as conventional farmed fish to collect mother populations rich in genetic diversity (see Table 1). Natural products were collected and collected in 3-5 year old mature mothers from the East Sea (East Coast), Taean and Buan (West Coast) and Geoje Island (South Coast) to adapt and tamper with indoor tanks. The eggs were collected by fertilized egg producers into different mothers.

As a result of measuring the total length, height and weight of 389 wild flounder and 735 cultured flounder of the mother population collected in the specific example of the present invention (see Table 1), the average ratio of body weight to obesity and total length was increased It was significantly higher than that (P <0.01) (see Table 2). In addition, after analyzing the genotype of the mother population and analyzing the genetic diversity, the natural population and the cultured flounder mother population collected for breeding showed that the genetic diversity in allele number, heterozygosity rate and allele frequency was increased. Abundant populations were identified (see Table 3). Next, genetic and genetic relationships of females and males were analyzed in order to draw crossbreeding instructions, and individuals with low mutual genetic flexibility and excellent growth and body phenotypes were selected for mating prior to breeding. Rank was determined. As a result, a mating guideline was prepared by a total of 762 parental candidates of 149 wild females, 371 wild females, 92 wild males and 150 wild males (see Table 5). Based on the mating guidelines, on April 19 and 28, 2005, 147 households and 181 households were produced through artificial insemination, and 245 households were produced except for duplicate households. To evaluate the genetic capacity of each F1 producing family, the paternity of each F1 individual was confirmed. As a result of analyzing each of the F1 individuals, 6,435 (95.6%) were identified as paternity, and 218 (89.0%) out of 245 artificially fertilized (see Table 7). As a result of analyzing the genetic diversity in the 2,874 F1 flounder, the genetic diversity of the F1 flounder improved compared to the conventional farmed mother population by including wild flounder in the mother group (see Table 8). In addition, the body weight index, obesity degree, heredity and breeding value of the body weight, total length, body length, height, head length, head length, length of disease, and length of the F1 individuals were analyzed. Phenotypic and genetic correlations of both were found to be high, suggesting that only one trait of weight, total length, and body weight could be improved with other traits (see Tables 9-13). In addition, as a result of experiments on the breeding effect of the body type by improving the height ratio for the total length, it was confirmed that the weight: height / height weight 70:30 is the best breeding effect of weight and body type (Fig. 2, Table 14 and Table 15).

In addition, in the specific example of the present invention, the female D1-0138 and the male D1-0103, the tenth-ranked females in the CK-0042 × CJ-0115 household, which are the first-ranked households based on the estimation result of the breeding price of the F1 individual, CK-0181 × CJ0114 ranked 33th in the household, female W1-0327 ranked 5th in the individual and male W1-0886 ranked 26th in the household, and CJ-0009 × CJ-0168 ranked 5th in the household ranked 5th in the household Six F1 individuals of female W1-1059 and fourth male W1-0017 were selected and inbred to produce breeding flounder of C7-1, C7-2 and C7-3 (Table 16, Table 17 and Figures). 1). In the growth efficiency comparison, the breeding flounder of the present invention was confirmed that the growth is faster compared to the cultured flounder and wild flounder (see Fig. 3). As a result, the breeding flounder produced by using the breeding method of the present invention was confirmed that the growth is faster than the conventional farmed and natural flounder, the method of breeding the flounder through selection breeding from the mother population having the genetic diversity of the present invention May be useful for improving the economics and productivity of flounder farming.

Step 1) is preferably performed through the size analysis of the flounder microsatellite markers amplified by any one or more of the following primer pairs, but is not limited thereto. Gene markers and primers for the flounder genotyping in the art If you have a pair, you can use them all:

Iii) a primer pair consisting of oligonucleotides each having SEQ ID NOs: 9 and 10 capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 1,

Ii) a primer pair consisting of oligonucleotides each having SEQ ID NOs: 11 and 12 capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 2,

Iii) a primer pair consisting of oligonucleotides each having SEQ ID NO: 13 and 14 capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 3,

Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 15 and 16, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 4,

Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 17 and 18, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 5,

Iii) a primer pair consisting of oligonucleotides each having SEQ ID NOs: 19 and 20 capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 6,

Iii) a primer pair consisting of oligonucleotides each having SEQ ID NO: 21 and 22 capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 7, and

Iii) A primer pair consisting of oligonucleotides having SEQ ID NOs: 23 and 24, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 8.

The expression trait of step 4) is body weight, full length and / or body weight, and may further measure and use one or more traits selected from the group consisting of body length, head length, head length, and head length.

In addition, the paternity of step 7) is preferably confirmed by analyzing the alleles of each progeny individual and comparing them with the alleles of the mothers, and then excluding the mothers that are in violation of Mendel's genetic law. Any method used in the industry to identify paternity can be used.

In addition, the breeding value for each trait of step 8)

Iii) measuring the weight, full length, body length, height, head, tail disease and tail length of the F1 flounder;

Ii) estimating heritability and genetic correlation for each trait; And,

Iii) can be measured by estimating individual breeding values for each trait, but is not limited thereto.

The heritability and genetic correlation is REMLF90 (Misztal, I., REMLF90 Manual, //nce.ads.uga.edu/~ignacy/numpub/blupf90/docs/remlf90.pdf) software, the breeder is BLUPF90 (Misztal, I ., BLUPF90, //nce.ads.uga.edu/~ignacy/numpub/blupf90/docs/blupf90.pdf), but it is preferred to estimate this, but is not limited and includes flounder used in the art. Any method can be used to estimate the heritability, genetic correlation or breeder value of farmed fish or livestock.

The present invention also provides a breeding flounder improved growth efficiency breeding by the above method.

The breeding flounder of the present invention has a growth rate of 20% or more, preferably 24% or more, as compared to conventional cultured flounder.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Collection of breeding flounder mother population

In order to secure genetic diversity of flounder mothers for breeding, natural flounder as well as conventional farmed fish were collected (Table 1). Among the wild products, Donghaeansan was collected from the East Sea, Westansan from Taean and Buan, and Namhaeansan was collected from mature mothers over 3-5 years, and the indoor tank was adapted and tamed. The producer collected them in different mothers. Among the collected mothers, morphological malformations such as eye position on the right side of the body side, gill cap malformation, dorsal body color on the white side, and blackening on the belly side in the wild were excluded. As a result of the body measurements of the mothers, the average ratio of obesity and height to total length was significantly higher than that of wild animals (P <0.01) (Table 2).

group Collection place Collection date Number of objects Measurement quality (mean ± standard deviation) Full length (cm) Height (cm) Weight (g) Natural East Coast (East Coast) December 2003 117 46.3 ± 5.3 15.6 ± 1.8 1,019 ± 379 West Coast (Tae'an) November 2003 46 43.8 ± 6.0 14.7 ± 2.3 895 ± 533 West Coast (Buan) November 2003 123 42.1 ± 4.0 13.9 ± 1.5 745 ± 295 South Coast (Geoje) May 2004 103 58.1 ± 7.9 20.1 ± 2.9 2,217 ± 920 sub Total 389 47.9 ± 8.7 16.2 ± 3.3 1,249 ± 835 Aquaculture Form 1 December 2003 187 51.6 ± 5.7 19.5 ± 2.6 1,845 ± 770 Form 2 December 2003 244 52.4 ± 6.2 18.8 ± 2.7 1,897 ± 751 Form 3 November 2003 181 44.1 ± 5.8 16.1 ± 2.8 1,121 ± 526 Form 4 May 2004 123 38.1 ± 1.3 13.3 ± 0.6 631 ± 70 sub Total 735 45.9 ± 7.8 16.7 ± 3.4 1,306 ± 780 Sum 1124 46.7 ± 8.2 16.5 ± 3.3 1,284 ± 801

※ Total length, body height, body weight

group   Collection place Measurement quality (LSMeans ± SE) Obesity Height / Battlefield (%) Weight / full length (g / cm) Body length / battlefield (%) Natural East Coast (East Coast) 9.9 ± 0.12 33.7 ± 0.13 21.4 ± 0.74 84.8 ± 0.12 West Coast (Buan)  9.6 ± 0.12 33.1 ± 0.13 17.3 ± 0.72 85.0 ± 0.12 West Coast (Tae'an) 9.84 ± 0.19 33.6 ± 0.21 19.5 ± 1.17 84.5 ± 0.19 Southern coast 11.8 ± 0.74 35.5 ± 0.85 35.9 ± 4.61 84.8 ± 0.74  sub Total 10.4 ± 0.16 34.1 ± 0.18 26.2 ± 0.99 85.1 ± 0.16 Aquaculture Form 1 12.4 ± 0.09 37.5 ± 0.11 34.4 ± 0.58 85.0 ± 0.09 Form 2 12.3 ± 0.13 35.8 ± 0.15 35.0 ± 0.80 85.2 ± 0.13 Form 3 12.1 ± 0.09 36.4 ± 0.11 24.4 ± 0.60 84.8 ± 0.10 Form 4 11.4 ± 0.09 35.0 ± 0.11 16.6 ± 0.58 86.2 ± 0.09  sub Total 12.1 ± 0.05 36.2 ± 0.06 27.6 ± 0.32 85.3 ± 0.05

Example  2. Genotype of flounder mother population genotype ) analysis

Microsatellite markers were used to investigate genotypes of flounder mothers for breeding. Microsatellite markers of K1 to K8 used in the analysis (Table 3, JH Kang et al., 2008, Int J Biol Sci , 4 (3): 143-149) and PCR primers (Table 4) designed to label three types of fluorescent dyes, FAM, NED, and HEX to amplify and distinguish these markers, and PCR conditions, etc. Korean Patent Publication No. 10-2009-0069898 was followed.

<2-1> flounder mother DNA  extraction

In order to extract genomic DNA from 389 wild flounder and 735 aquaculture flounder of the collected mother population, a part of the flounder pectoral fin tissue was cut out. At this time, RFID (Radio Frequency IDentification) tag (cover) was inserted in the back muscle so that each individual could be distinguished by the unique number of alphabet and number combination read through the reader. The dorsal tissue was cut by about 1 cm of the softened area except for the low-thickness area to facilitate regeneration after cutting, and TNES-Urea buffer solution (10 mM Tris-Cl; pH 7.5, 125 mM NaCl, 10 mM EDTA, 1% SDS and 6). M urea containing) 600 μl, protease K (proteinase K, Sigma, USA) was treated at a final concentration of 100 ㎍ / ㎖ and dissolved at 37 ℃ overnight. The same amount of phenol / chloroform / isopropanol (Sigma, USA) was added to the sample, shaken, and centrifuged at 12,000 rpm for 1 minute, and then the upper layer was collected twice to obtain a supernatant containing DNA. 100% ethanol, twice the amount of DNA solution, was centrifuged at 12,000 rpm for 30 seconds, and the precipitate was collected. The excess salt was washed with 70% ethanol, followed by TE (10 mM Tris-Cl; pH 8.0, 1 mM). EDTA) was dissolved in the buffer solution and stored refrigerated. The DNA was used for the following genetic analysis.

Seat
(Locus)
SEQ ID NO: Repeat motif Repeated center size
(Size range of repeat motifs; bp) Genbank
accession no. K1 One (CA) ₆ CG (CA) ₁₄ 42 AY328958 K2 2 (CT) ₁₄ AT (CT) ₄ 38 AY328977 K3 3 (TC) ₂₄ 48 AY328973 K4 4 (CT) ₅ CA (CT) ₁₄ 40 AY328982 K5 5 (GT) ₅ AT (GT) ₅ AA (GA) ₄ TT (GA) ₃ AA (GA) ₅ TT (GA) ₆ GTAA (GA) ₃ GT (GA) ₁₈ 114 AY328981 K6 6 (GACA) ₂ (CA) ₁₀ 28 AY328959 K7 7 (CT) ₆ CA (CT) ₁₄ (GT) ₆ 54 AY328976 K8 8 (AC) ₅ AA (CA) ₁₇ AT (AC) ₂ TC (TG) ₂ TTT (TG) ₉ 79 AY328965

Marker SEQ ID NO: Primer sequence (5 '→ 3') K1 9 F: GCG ACT CTC AAA AAT CCG TCA G 10 R: GGA CTT TGT GGG GTT TCA GGA K2 11 F: GTG TAT GTG TTT CGT TTC CTG C 12 R: TGA AGG CTG TTG TTG CTG TAG K3 13 F: GAA GGA TCT GGA CTC ATG GTG AC 14 R: CAG CAG CAA AGG CAG AAA GAG K4 15 F: TCC TTC ACA CTA ATG GAG TCC A 16 R: GTG TCC GAA AGG AAA ACT AGG K5 17 F: ACA GTA AAA CAG TCC CTC CTG AAC 18 R: AGG AGT CTG GAA CCA AAT GTC TG K6 19 F: AGA GGA TAT CGA GGG GAG G 20 R: CAG CAG TAG CCG ATC TTA GTG K7 21 F: TTG AAA CTG GAT GCC TCA C 22 R: TCT CAC GTT TCC ATG TCA G K8 23 F: ATC AGG AAC GGG AGA GAC TC 24 R: GAG TTC GTC CTT CGT CTT CA

<2-2> olive flounder Mother-group  Genetic Diversity Analysis

Genetic amplification reaction according to the Republic of Korea Patent Publication No. 10-2009-0069898 using DNA obtained from the flounder pectoral fin tissue in Example 2-1 as a template DNA genotype analyzer (3130 genetic analyzer) ABI, USA) was used to analyze genotypes represented by peaks. Diversity analysis of genotypes is described in Cervus 2.0 (http://helios.bto.ed.ac.uk/evolgen/cervus/cervus.html) and MSA (Microsatellite Analyzer; Dieringer D & SchlC, 2003, Mol Ecol Notes 10: 1046-1048) were used to analyze allele number, size, frequency, heterozygosity, and polymorphism index.

The number of alleles in the flounder mother population ranged from 5 to 33 according to the markers. The average number of aquatic mothers was 17, while the wild mothers had an average of 24.5 alleles. In addition, the expectation of heterozygosity rate showed that the wild mothers were 0.692 ~ 0.950 depending on the position of the markers, which is much more diverse than the 0.599 ~ 0.899 of the cultured fish (Table 5). Genetic diversity in populations, heterozygotes, and allele frequencies was identified as abundant in genetic diversity.

Seat division Group (by collection place) Natural Aquaculture all East coast West coast South coast Form A Form B Form C Form D K1 N 117 169 103 187 244 181 123 389 735 1124 A 24 27 26 14 11 12 14 30 16 30 Ho 0.923 0.893 0.893 0.775 0.848 0.823 0.870 0.902 0.827 0.853 He 0.926 0.910 0.930 0.804 0.854 0.802 0.829 0.922 0.830 0.876 PIC 0.917 0.900 0.920 0.783 0.836 0.781 0.808 0.915 0.814 0.865 Fis 0.001 0.016 0.037 0.034 0.005 -0.028 -0.051 0.019 -0.004 0.005 K2 N 117 169 103 187 244 181 123 389 735 1124 A 19 19 20 15 16 14 15 21 16 22 Ho 0.872 0.888 0.806 0.941 0.902 0.895 0.870 0.861 0.905 0.890 He 0.924 0.919 0.920 0.904 0.883 0.877 0.874 0.923 0.894 0.910 PIC 0.914 0.911 0.910 0.893 0.871 0.863 0.858 0.917 0.884 0.903 Fis 0.054 0.033 0.123 -0.043 -0.022 -0.022 0.003 0.065 -0.022 0.009 K3 N 117 169 103 187 244 181 123 389 735 1124 A 20 22 19 16 14 15 15 26 16 26 Ho 0.923 0.888 0.893 0.893 0.844 0.901 0.846 0.900 0.871 0.881 He 0.909 0.919 0.916 0.889 0.846 0.899 0.863 0.915 0.883 0.907 PIC 0.898 0.910 0.905 0.877 0.831 0.888 0.847 0.908 0.873 0.900 Fis -0.017 0.033 0.023 -0.006 0.002 -0.003 0.019 0.017 0.003 0.008 K4 N 117 169 103 187 244 181 123 389 735 1124 A 33 32 32 15 23 16 17 38 27 39 Ho 0.949 0.893 0.951 0.882 0.844 0.851 0.846 0.925 0.856 0.880 He 0.938 0.925 0.950 0.806 0.858 0.790 0.850 0.947 0.833 0.893 PIC 0.930 0.925 0.943 0.782 0.842 0.763 0.832 0.944 0.816 0.884 Fis -0.014 0.033 -0.004 -0.096 0.015 -0.079 0.003 0.011 -0.035 -0.018 K5 N 117 169 103 187 243 181 123 389 734 1123 A 26 25 26 19 20 17 16 30 23 30 Ho 0.897 0.964 0.913 0.882 0.889 0.895 0.878 0.931 0.887 0.902 He 0.918 0.924 0.922 0.890 0.892 0.899 0.882 0.925 0.898 0.920 PIC 0.908 0.924 0.912 0.877 0.881 0.887 0.866 0.919 0.889 0.914 Fis 0.020 -0.046 0.008 0.007 0.003 0.003 0.002 -0.010 0.005 0.000 K6 N 117 169 102 186 244 181 123 388 734 1122 A 6 7 10 5 6 5 5 10 6 10 Ho 0.701 0.669 0.667 0.656 0.713 0.635 0.699 0.678 0.677 0.677 He 0.701 0.692 0.711 0.682 0.695 0.599 0.749 0.711 0.698 0.710 PIC 0.645 0.629 0.656 0.621 0.639 0.546 0.701 0.654 0.645 0.656 Fis -0.002 0.033 0.060 0.036 -0.027 -0.062 0.064 0.032 0.000 0.011 K7 N 117 165 103 185 243 181 108 385 717 1102 A 19 20 22 16 15 13 14 23 16 23 Ho 0.880 0.909 0.922 0.795 0.872 0.762 0.796 0.901 0.813 0.844 He 0.930 0.910 0.926 0.773 0.840 0.769 0.765 0.925 0.809 0.872 PIC 0.921 0.910 0.916 0.756 0.820 0.744 0.743 0.919 0.790 0.861 Fis 0.051 0.000 0.002 -0.029 -0.039 0.008 -0.044 0.018 -0.025 -0.008 K8 N 99 150 99 174 236 179 99 348 688 1036 A 16 17 16 14 12 10 15 18 16 18 Ho 0.798 0.767 0.828 0.884 0.708 0.782 0.798 0.793 0.785 0.788 He 0.897 0.867 0.892 0.828 0.748 0.840 0.889 0.883 0.827 0.850 PIC 0.883 0.867 0.877 0.804 0.723 0.819 0.874 0.872 0.808 0.835 Fis 0.108 0.115 0.069 -0.069 0.053 0.067 0.100 0.102 0.034 0.058 Average A 20.38 21.13 21.38 14.25 14.63 12.75 13.88 24.5 17 24.75 He 0.893 0.883 0.896 0.822 0.827 0.809 0.838 0.894 0.834 0.867 PIC 0.877 0.867 0.88 0.799 0.805 0.786 0.816 0.881 0.815 0.852

N: population, A: allele, H (o): Observed heterozygosity, H (e): Expected heterozygosity, PIC: Polymorphic information content, Fis : Inbreeding coefficient.

Example  3. Preparation of mating guidelines for household production

In order to develop the mating guidelines, the genetic genetic relationship with male individuals was calculated by using the mutual genetic genetic relationship matrix with female mothers. Specifically, genetic flexibility between each subject was calculated by genetic genetic relation algorithm (Queller DC and Goodnight KF, 1989, Evolution 43: 258-275) from the microsatellite marker genotype data of each subject.

When the alleles for the N markers for the subject y were compared with the subject x as shown in Table 6, statistical predictions about the genetic flexibility between the two subjects were calculated using Equation 1 below.

Locus One 2 … i … 2N  x (Reference individual) a b  y (Proband individual) c d

: Genetic relationship between x and y entities

P: frequency of the i allele of subject x

Px: 1 if the i allele of x is homozygous, 0.5 if heterozygous (i = even if i = even, 1 if equal to i-1, 0.5 if different; i + 1 if i = odd 1 if equal, 0.5 if different)

Py: The i allele of x is 0 compared to the two alleles of y and 0 if there is no equal, 0.5 if there is one, and 1 if there are two equals (i = i-allele of x and i- of y Compare 1 and i alleles; if i = odd, compare i alleles of x and i and i + 1 alleles of y)

Each male mother was screened for males with a genetic distance of less than 0.2 by calculating the genetic genetic relationship, and the males with excellent growth and body phenotypes were selected from 1st to 3rd place. A mating priority was created. As a result, mating guidelines were prepared by a total of 762 parental candidates, 149 wild females, 371 aquaculture females, 92 wild males and 150 aquaculture males (Table 7).

Example  4. Breeding household production by artificial insemination

On the basis of the mating guidelines prepared from the mixed populations of cultured flounder and wild flounder, 147 households and 181 households were produced through artificial insemination on April 19 and 28, 2005, among which duplicate households were excluded. A total of 245 households were produced. Specifically, the male and female sperm were extracted by the abdominal compression method while paying attention to the male and female pairs on the mating instructions through the RFID markers inserted into the individual mothers, so that no foreign substances such as seawater and excreta were mixed. Next, the artificial sperm and the egg were mixed with the egg and pour the sea water to activate the sperm and artificially inseminated for 2 minutes, the excess sperm was washed with sea water. The fertilization rate of artificially fertilized households was calculated at 2 to 3 hours after artificial fertilization, and fertilized eggs were mixed in a tank to contain 5000 eggs per household. At this time, the water temperature in the tank for egg development was maintained at 18 ℃, 20 ℃ until 60 days after incubation, 18 ℃ until 70 days, and then raised in natural water temperature. The illuminance was adjusted to 300 Lux and the photoperiod from 08:00 to 20:00, and the feeding density and breeding density in the tank were bred under the conditions shown in Table 8.

Breeding date and time after hatching (Sun) Food type, feeding rate / day 3-12 2 rotifers 13-17 Rotifer, Altemia 2 each 18-28 Rotifer, Altemia 2 times, Initial Feed No. 2 3 times 29-34 Altemia 3 times, Early Feed 3 No. 6 35-41 Initial Feed 4 No. 6 42-53 Initial Feed No. 5 Five Times 54-59 Initial Feed 6 No. 4 60-64 Initial Feed No. 7 4 times 65-94 Compound feed No. 1 4 times 95-127 Compound feed No. 2 4 times 128-157 Compound feed three times four times 158-174 Compound feed four times three times 175-219 Compound feed No. 5 3 times 220 or more Compound feed six times three times

Example  5. Genotyping of F1 Flounder

<5-1> Parental Identification for Household Genetic Evaluation

To evaluate the genetic capacity of each F1 family, paternity was performed to find the parents of each individual. For each of 6,731 F1 individuals produced during the second round, the dorsal tissue was cut out by inserting a wireless identification tag for identification of the individual into the back muscle at day 170, and then in the same manner as in Examples 2-1 and 2-2. DNA was extracted, polymerase chain reaction was performed, and genotypes of the respective markers were obtained by genotyping.

The allelic type of each F1 individual obtained is compared with the allelic type of the mothers, and the parent combination estimated by the exclusion method that violates Mendel's genetic law is compared with the artificially modified list by the mating instructions. The paternity was confirmed. As a result of analyzing 6,731 F1 individuals, 6,435 (95.6%) were identified as paternity, and 218 (89.0%) of 245 artificially fertilized (Table 9). At this time, the number of descendants of the paternity households varied from one confirmed per household to as many as 117 identified, and the unidentified households were naturally reduced or hatched during hatching and breeding. It is thought to be included in the population of inferior sharks that are culled by one growth difference.

Primary household production Second household production system Analysis 2,431 4,300 6,731 Pedigree 2,267 (93.3%) 4,168 (96.9%) 6,435 (95.6%) Production line 147 181 245 Household count 118 (80.3%) 157 (86.7%) 218 (89.0%)

 <5-2> Genetic Diversity Analysis of F1 Flounder

Analysis of genetic diversity of 2,874 F1 flounder using microsatellite markers revealed that the number of alleles was 20.38 and heterozygosity expected to be 0.840. Genetic diversity of F1 flounder has been improved (Table 10).

Aquaculture Mother Group F1 flounder  Analysis object count 735 2874  Allele number 17.00 20.38  Observation heterojunction (Ho) 0.828 0.831  Expected Release Hemolysis (He) 0.834 0.84  Polymorphism Index (PIC) 0.815 0.822

H (o): observed heterojunction, H (e): expected heterojunction, PIC: polymorphism index

Example  6. Measurement of Measurement Traits for Genetic Capacity Assessment

Measurement of traits such as total length, body height and body weight was performed to evaluate heritability in F1 flounder at day 170 after incubation, and body shape was determined by the ratio of the total length / height, The condition factor was evaluated by weight / length 3.

<6-1> Heredity and Breeder  calculation

REMLF90 (Misztal, I., REMLF90 Manual, 2002) software (//nce.ads.uga.edu/~ignacy/numpub/blupf90/docs/remlf90.pdf) using measurement data of F1 individuals at day 170 after hatching. Estimate the heritability and genetic correlation for each trait using BLUPF90 (Misztal, I., BLUPF90, 2008) software (//nce.ads.uga.edu/~ignacy/numpub/blupf90/docs/blupf90.pdf) Was used to estimate individual breeders for each trait.

Genetic parameters were estimated for 1,197 primary production groups and 1,571 secondary production groups for breeding flounder.

Weight (g) Full length (cm) Length (cm) Height (cm) Two pieces (cm) Armed Forces (cm) US illness (cm) Primary 93.1 ± 0.70 19.9 ± 0.05 16.7 ± 0.05 6.99 ± 0.021 4.58 ± 0.014 1.05 ± 0.004 1.84 ± 0.006 Secondary 79.1 ± 0.61 19.4 ± 0.05 16.0 ± 0.04 6.69 ± 0.018 4.50 ± 0.013 0.96 ± 0.003 1.70 ± 0.005

※ Two pieces-Length from the tip of the fish snout to the tip of the gill cap.

※ The minimum height of the US Army-US Army (US Army: The area from the base of the last softening of the dorsal fin to the base of the caudal fin)

※ Length from base of last disease-dorsal fin to caudal fin base

Obesity Body length / battlefield Height / Battle Head / Full Length U.S. Army / Battlefield Armed Forces / Battlefield Primary 11.41 ± 0.023 83.69 ± 0.032 35.08 ± 0.043 23.03 ± 0.039 9.23 ± 0.017 5.28 ± 0.016 Secondary 10.64 ± 0.020 82.78 ± 0.028 34.53 ± 0.038 23.23 ± 0.034 8.79 ± 0.015 4.98 ± 0.014

In addition, the present inventors compared the growth of each household by making a ranking for each household based on the average weight of each family of flounder flounder used for genetic parameter estimation (Table 13).

Primary Secondary Breeding parent (male and female) Average weight (g) ranking Breeding parent (female X male) Average weight (g) ranking CA036-CJ055 138.9 One CH003-CJ181 117.5 One CK181-CJ114 121.2 2 CJ048-WT030 111.8 2 WD035-CJ052 115.0 3 CA059-CJ167 111.4 3 CH017-CJ067 114.7 4 WB092-CH073 109.0 4 CA036-CJ066 112.7 5 CA006-CA076 107.7 5 CH023-CJ066 112.0 6 WB034-CJ056 105.0 6 CH001-WT034 110.5 7 CH026-CA148 104.0 7 CJ009-CJ168 110.4 8 CK106-CJ095 102.6 8 CA015-CJ146 110.0 9 CK029-CJ137 102.3 9 CH018-CJ142 108.9 10 CA093-CJ161 101.7 10 : : : : : : WD015-CJ016 41.0 101 CH020-WG024 47.8 148 WD090-WD057 39.0 102 WD054-CA113 47.2 149 WG031-CJ046 36.0 103 WD023-WD027 42.5 150 WT028-WG009 36.0 103 WB003-CJ123 34.0 151 WG031-WT029 26.0 105 WT028-WG009 31.0 152

The heritability for the irradiation traits of sarcoma flounder (F1), weight, length, height, head length, disease length, disease length, obesity, and height / length is shown in Table 14, and phenotypic and genetic correlations are shown in Table 15. Phenotypic correlations between traits related to growth, weight, height, and height were 0.92-0.95, and 0.98, respectively. Therefore, weight, height, and height, which are highly related to growth, are highly correlated with each other. Therefore, improvement of only one trait may lead to improvement in other traits. We believe that this will be possible through the improvement of (Table 15).

weight Battlefield Height Two Untreated A soldier Obesity Height / Battle Heredity
(SE) 0.53
(0.057) 0.57
(0.060) 0.57
(0.059) 0.42
(0.053) 0.47
(0.055) 0.14
(0.031) 0.34
(0.046) 0.29
(0.043)

weight Battlefield Height Obesity Height / Battle weight - 0.95 0.93 0.38 0.23 Battlefield 0.98 - 0.92 0.13 0.10 Height 0.98 0.98 - 0.34 0.49 Obesity 0.46 0.02 0.45 - 0.56 Height / Battle 0.72 0.05 0.78 0.87 -

* Upper right: phenotypic correlation, lower left: genetic correlation

In addition, the breeding value range of -27.54 to 35.14 g for the weight of the mother flounder used in the breeding, and the breeding value of the first generation (F1) of breeding flounder (F1) was -37.80 to 56.91 g (Table 16).

Individual number division weight Battlefield Height Two Obesity Height / Battle Untreated A soldier 110006 Acquaintance -27.54 -2.44 -0.998 -0.523 -0.254 -0.048 -0.094 -0.607 110022 Acquaintance -24.17 -2.22 -0.817 -0.389 -0.174 -0.063 0.184 -0.180 110040 Acquaintance 31.03 2.12 0.653 0.458 0.208 0.025 0.157 -0.211 110099 Acquaintance 35.14 2.74 1.116 0.536 0.286 0.083 -0.016 0.698 110126 Acquaintance -26.86 -2.33 -1.019 -0.531 -0.231 -0.030 -0.311 -0.855 110136 Acquaintance -24.23 -2.28 -0.899 -0.466 -0.198 -0.038 -0.008 -0.431 110138 Acquaintance 29.63 2.06 0.830 0.390 0.232 0.067 0.277 0.414 120019 Acquaintance 33.32 2.11 0.770 0.528 0.259 0.036 0.308 -0.014 120063 Acquaintance -26.21 -1.97 -0.700 -0.445 -0.277 -0.056 -0.325 0.021 120103 Acquaintance 29.95 2.33 0.815 0.456 0.257 0.051 -0.102 0.014 210053 F1 49.19 2.97 0.927 0.763 0.341 0.047 0.396 -0.275 210080 F1 -36.42 -3.70 -1.407 -0.737 -0.310 -0.061 -0.124 -0.523 210130 F1 56.91 3.56 1.443 0.554 0.336 0.054 0.179 0.444 210133 F1 -37.80 -3.88 -1.596 -0.725 -0.348 -0.067 -0.094 -1.116 210414 F1 56.54 3.64 1.475 0.848 0.388 0.067 0.119 0.461 210506 F1 -34.52 -3.05 -1.203 -0.714 -0.308 -0.060 -0.357 -0.730 210656 F1 -31.60 -2.89 -1.084 -0.577 -0.244 -0.047 -0.054 -0.545 210710 F1 -34.88 -3.25 -1.175 -0.612 -0.308 -0.050 -0.195 -0.427 210901 F1 56.54 3.33 1.411 0.041 0.412 0.038 0.361 0.562 211001 F1 -33.55 -3.17 -1.183 -0.606 -0.307 -0.067 -0.220 -0.250 211019 F1 49.98 3.56 1.225 0.608 0.348 0.049 -0.083 0.075 211188 F1 49.98 3.17 1.245 0.667 0.360 0.054 0.220 0.360 211213 F1 -31.89 -2.67 -0.903 -0.531 -0.356 -0.064 -0.381 0.097 211418 F1 52.90 3.45 1.300 0.576 0.382 0.056 0.139 0.293 211451 F1 48.89 3.05 1.308 0.485 0.311 0.035 0.280 0.562 220297 F1 -32.96 -3.58 -1.418 -0.674 -0.333 -0.056 0.063 -0.708 :
: :
: :
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: :
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: :
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:

In addition, when selecting the growth and body shape at the same time, the growth trait and body shape such as weight, length, and height are negative (-) correlation, the body shape worsens only considering growth, and conversely, the body / body length ratio If it is lowered to 34.1, the growth effect is very small. Thus, in order to evenly improve the two opposing traits, the estimated breeding value is used for 50:50, 60:40, 70:30, and 80:20 for body weight and body traits, respectively. The selection index was calculated as follows.

선발지수 =

Selection Index =

Where: standardized value of weight breeders,

        : Standardized value of body breeders,

 : Values of the breeding values of the traits were normalized so that the total mean was 0 and the standard deviation was ± 1.

Where: breeder of the trait

        : Mean of motive group of traits

        : Standard deviation of motive group of traits

       Motivational group: whole entity used for analysis

As a result of predicting the breeding effect of the next generation according to each weight using Equation 2, the breeding effect of the growth trait of weight, total length, height and obesity was the highest at 80:20. However, the breeding value of body type (height / length) was the highest at 50:50. Thus, the inventors determined that the weight and body weight breeding effect is the best when the weight of body weight / height / length is 70:30 in which growth is slowed in consideration of both the growth and body breeding effects (FIG. 2 and Table 17).

Weight (weight: height / battlefield) Weight (g) Full length (cm) Height (cm) Obesity Height / Battle 50: 50 104.27 21.11 7.23 10.96 34.40 60: 40 107.85 21.32 7.35 11.03 34.56 70: 30 110.08 21.44 7.44 11.09 34.72 80: 20 111.31 21.51 7.49 11.13 34.85 F1 average ability  85.28 19.61 6.82 10.99 34.76

Example  7. Production of growth and body improvement household

It is preferable to select the top households and individuals based on the result of the genetic ability evaluation, but because genetic homology requires crossbreeding between males and females in the family, the top households containing all the males and females among the selected individuals are selected. CK-0042 × CJ-0115, the No. 1 household ranking, female D1-0138 with the highest individual ranking in the household, male D1-0103 with the 10th largest ranking, and CK-0181 × CJ0114, ranked No. 5 in the household ranking 33th Six F1 individuals were selected: W1-0327 and male W1-0886, 26th, and CJ-0009 × CJ-0168, the fifth-largest household, female W1-1059 and 35th male W1-0017, respectively. Inbreeding was then performed to produce C7-1, C7-2 and C7-3 genetic net lineages (Tables 18, 19 and

ancestry Individual number Weight (670 days) Ranking mother part Household ranking C7-1 mother D1-0138 3841 One CK-0042 CJ-0115 One part D1-0103 1711 10 C7-2 mother W1-0327 3244 5 CK-0181 CJ-0114 33 part W1-0886 1665 26 C7-3 mother W1-1059 2749 35 CJ-0009 CJ-0168 5 part W1-0017 2017 4

Object number gender K1 K2 K3 K4 K5 K6 K7 K8 F0 CK-0042 cancer 84 112 162 164 222 226 98 128 179 195 113 117 139 141 186 198 CJ-0115 Number 82 98 170 170 204 208 124 132 187 197 113 115 141 143 184 190 CK-0181 cancer 98 98 172 172 198 202 128 146 177 195 115 115 141 141 182 186 CJ-0114 Number 94 102 152 172 204 210 118 118 185 203 115 117 155 167 190 204 CJ-0009 cancer 84 98 146 170 220 226 128 128 179 183 113 115 141 141 184 198 CJ-0168 Number 84 98 156 166 214 222 98 128 177 197 113 121 141 159 190 200 F1 D1-0138 cancer 84 98 164 170 204 226 124 128 187 195 113 113 141 143 186 190 D1-0103 Number 82 84 164 170 208 222 124 128 195 197 115 117 141 143 184 198 W1-0327 cancer 98 102 152 172 198 210 118 128 177 185 115 115 141 167 182 190 W1-0886 Number 94 98 172 172 198 204 118 128 185 195 115 117 141 155 186 190 W1-1059 cancer 98 98 146 156 220 222 128 128 177 179 113 113 141 141 184 200 W1-0017 Number 84 98 166 170 222 226 98 128 183 197 113 115 141 159 198 200

Example 8 Growth Efficiency Assay of Breeding Flounder

In order to test the growth efficiency of the second generation flounder produced in Example 7, the comparison was made with seedlings produced from cultured flounder and wild flounder. At this time, the farmed flounder was naturally spawned from 15 females (average weight 2430 g) and 18 males (average weight 1130 g), and the wild flounder was 20 females (average weight 2560) and 18 males (average weight 1260 g). Seeds produced using fertilized eggs were used. Breeding flounder at 215 days after hatching had an average weight of 322.7 g, which was 25.2% faster than 257.8 g of cultured flounder and 120.5% faster than 146.2 g of wild flounder, and average weight of breeding flounder at 12 months after seeding. Was 886.1 g, which showed 24% faster growth than the average weight of 714.5 g of cultured flounder at the same time (Figure 3).

Figure 1 is a schematic diagram of the production of growth and body type breeding flounder flounder family.

Figure 2 is a photograph of the first generation flounder breeding growth and body shape.

Figure 3 is a diagram showing the growth efficiency test results of growth and body type breeding second generation flounder.

<110> National Fisheries Research and Development Institute <120> Breeding method for genetically improved olive flounder which          grows faster <130> 9p-10-01 <160> 24 <170> Kopatentin 1.71 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> K1 <400> 1 cacacacaca cacgcacaca cacacacaca cacacacaca ca 42 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> K2 <400> 2 ctctctctct ctctctctct ctctctctat ctctctct 38 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> K3 <400> 3 tctctctctc tctctctctc tctctctctc tctctctctc tctctctc 48 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> K4 <400> 4 ctctctctct cactctctct ctctctctct ctctctctct 40 <210> 5 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> K5 <400> 5 gtgtgtgtgt atgtgtgtgt gtaagagaga gattgagaga aagagagaga gattgagaga 60 gagagagtaa gagagagtga gagagagaga gagagagaga gagagagaga gaga 114 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> K6 <400> 6 gacagacaca cacacacaca cacacaca 28 <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> K7 <400> 7 ctctctctct ctcactctct ctctctctct ctctctctct ctgtgtgtgtgt gtgt 54 <210> 8 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> K8 <400> 8 acacacacac aacacacaca cacacacaca cacacacaca cacacaatac actctgtgtt 60 ttgtgtgtgt gtgtgtgtg 79 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K1 forward primer <400> 9 gcgactctca aaaatccgtc ag 22 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K1 reverse primer <400> 10 ggactttgtg gggtttcagg a 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K2 forward primer <400> 11 gtgtatgtgt ttcgtttcct gc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K2 reverse primer <400> 12 tgaaggctgt tgttgctgta g 21 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> K3 forward primer <400> 13 gaaggatctg gactcatggt gac 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K3 reverse primer <400> 14 cagcagcaaa ggcagaaaga g 21 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K4 forward primer <400> 15 tccttcacac taatggagtc ca 22 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K4 reverse primer <400> 16 gtgtccgaaa ggaaaactag g 21 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> K5 forward primer <400> 17 acagtaaaac agtccctcct gaac 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> K5 reverse primer <400> 18 aggagtctgg aaccaaatgt ctg 23 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K6 forward primer <400> 19 agaggatatc gaggggagg 19 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K6 reverse primer <400> 20 cagcagtagc cgatcttagt g 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K7 forward primer <400> 21 ttgaaactgg atgcctcac 19 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K7 reverse primer <400> 22 tctcacgttt ccatgtcag 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> K8 forward primer <400> 23 atcaggaacg ggagagactc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> K8 reverse primer <400> 24 gagttcgtcc ttcgtcttca 20  

Claims (5)

1) extracting DNA from pectoral fin tissue of the flounder female population and the male population; 2) analyzing the size of the flounder microsatellite markers amplified using at least one primer pair from the DNA of each population extracted in step 1):   Iii) a primer pair consisting of oligonucleotides each having SEQ ID NOs: 9 and 10 capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 1;   Ii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 11 and 12, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 2;   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 13 and 14, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 3;   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 15 and 16, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 4;   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 17 and 18, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 5;   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 19 and 20, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 6;   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 21 and 22, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 7; And,   Iii) a primer pair consisting of oligonucleotides having SEQ ID NOs: 23 and 24, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 8; 3) calculating genetic flexibility between the individual of the female group and the male group according to Equation 1 below; &Quot; (1) &quot;

 : Genetic relationship between x and y entities   P: frequency of the i allele of subject x   Px: 1 if the i allele of x is homozygous; 0.5 if heterozygous 1 if equal, 0.5 if different)   Py: The i allele of x is 0 compared to the two alleles of y and 0 if there is no equal, 0.5 if there is one, and 1 if there are two equals (i = i-allele of x and i- of y Compares 1 and i alleles; if i = odd, compares i allele of x and i and i + 1 allele of y) 4) selecting a male individual of the female population of the female population and a male population of which the calculated genetic relationship is 0.2 or less; 5) selecting an individual whose expression trait of any one of body weight or full length is the top 1 to 3 of the male individuals selected in step 4); 6) mating the male individual selected in step 5) and the female individual of step 4) through artificial insemination; 7) breeding the F1 generation produced by the crossing of step 6) for 150 to 200 days; 8) confirming paternity of the F1 generation; 9) measuring breeding value using the weight, total length, body length, height, head length, uncompensated disease, and untreated disease values of F1 individuals whose parental relationship of step 8) is confirmed; 10) weight of the household produced as a result of the mating of step 6): selecting female and male individuals in the household ranking when the weight / length of the weight is 70:30 and inbreeding; And, 11) A breeding method for enhancing the growth efficiency of the flounder comprising the step of assaying the growth efficiency of the breeding flounder produced by the cross of step 10). delete The method according to claim 1, wherein the expression trait of any one of length, head, head disease, or head disease can be additionally measured and used as an indicator in step 5). According to claim 1, wherein the breeding value of step 9) is  Iii) measuring the weight, total length, body length, height, head length, head disease, and disease length of the F1 flounder;  Ii) measuring the heritability and genetic correlation for each trait using the values measured in step iii); And,  Iii) measuring the individual breeding value for each trait using the value measured in step iii). delete

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