Cytogenetic Analysis of All-Female Triploid Olive Flounder Paralichthys olivaceus for Ploidy Verification

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한수지 49(5), 671-674, 2016

671 Copyright © 2016 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

Korean J Fish Aquat Sci 49(5),671-674,2016

Short Communication

넙치

(Paralichthys olivaceus)

는극동 지역에서양식되고 있 는해산어종으로서우리나라에서는국립수산과학원육종센터 에서선발육종기법으로

‘

^킹넙치

(KingNupchi)’

^를 ^{생산하였고}

, 2013

^년도부터

golden seed project (GSP)

^사업을^통해^킹넙치 의불임종자를수출하기위한연구를수행해오고있다

(Jung et al., 2014).

그러나불임종자인

3

배체는그유도율이

90%

이상 이어야산업적이용이가능함을보고한바있어

3

^배체를^유도 한후그집단의배수체빈도측정은유도된불임종자의산업적 이용에필수요건이다

(Thorgard, 1986).

이에본연구에서는킹넙치의불임화를위해수정란을저온 처리하여전암컷

3

배체를유도한후양식현장에서쉽고간편 한

3

^배체^분석법을^{보급하고자}^다양한^{세포유전학적}^분석방법 들을비교검토하였다

.

전암컷

3

^배체^킹넙치^유도를^위해

3-4

^년생^대조군^넙치^암컷 을손으로복부를압박하여난을확보하였으며

, Kang (2016)

^의 방법으로유도된자성발생성수컷킹넙치의

4

가계

(0219, 0335, 1697

^및

1788)

^및^대조군^수컷^넙치의^복부를^압박

,

^채정하여

수정

3

분후

2-4℃

로

45

분간저온처리하였다

. 1

회실험시각 실험군별로수정난

30-50 cc

^를^처리하여^각^{실험군에서}

110

^개 의난을

3

^회^추출하여^결과를^{도출하였고}

,

^이들^실험을

3

^회^반 복수행하였다

.

^각^실험군의^부화율과^부화^후^난황을^흡수하 지못하고죽는자어의빈도를측정한후

,

^생존율을^확인하였 다

.

종자생산은제주시우도수산에서

18-21℃

의수온조건으로 사육하였다

.

^단순하며^경제적인^넙치의

3

^배체^{분석방법을}^비교 검토하기위해세포및핵의크기

, flow cytometry,

^염색체^분 석

, Ag-NOR banding

^및^지느러미^{조직으로부터}

solid tissue technique (Klingerman and Bloom, 1977)

^에^의해^얻어진^세포 에대하여

silver staining

을수행하였다

.

대조군과

4

^가계의^전^암컷^넙치^실험군의^부화율

,

^비정상^개 체발생률및생존율에대한자료는

Table 1

^에^{나타내었다}

.

^부 화율에있어

2

^배체는^대조군과

‘0219’

^가계의^부화율이

75%

^이 상으로높게나타났으나

, ‘0335’, ‘1697’

^및

‘1788’

^가계들의^경 우

30.4-48.7%

로유의적으로낮게나타났다

(P<0.05).

그러나

‘0219’

^가계의

3

^배체^부화율은

26.0%

^로^대조군

(44.2%)

^및^나머

전 암컷 3배체 넙치(Paralichthys olivaceus)에 대한 효율적인 세포유전학 분석법

고민균·정효선·이효빈·김동수

¹

*

부경대학교 수산생물학과, ¹부경대학교 해양바이오신소재학과

Cytogenetic Analysis of All-Female Triploid Olive Flounder Paralichthys olivaceus for Ploidy Verification

Min Gyun Ko, Hyo Sun Jung, Hyo Bin Lee and Dong Soo Kim ¹ *

Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea

1

Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan 48513, Korea

We cytogenetically analyzed a triploid King-Nupchi strain of the olive flounder Paralichthys olivaceus to define the simplest, most rapid, and most effective method of ploidy analysis in aquaculture farms. Female triploidy of the flounder King-Nupchi strain was induced by cold shock (3 min post-fertilization at 2-4°C for 45 min). Triploid in- duction was confirmed by erythrocyte measurement (nuclear volume, 29.15±2.10 µm

³

); flow cytometry (2.14±0.03 pg/cell); chromosome count (3N=72); Ag-NOR banding; and silver staining. Silver staining of finned cells obtained using a solid tissue technique was the most effective method of ploidy verification.

Key words: KingNupchi, Paralichthys olivaceus , Triploid, Silver staining

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5657/KFAS.2016.0671 Korean J Fish Aquat Sci 49(5) 671-674, October 2016

Received 9 September 2016; Revised 21 October 2016; Accepted 22 October 2016

*Corresponding author: Tel: +82. 51. 629. 5914 Fax: +82. 51. 629. 5908

E-mail address: [email protected]

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고민균

ㆍ

정효선

ㆍ

이효빈

ㆍ

김동수

672

지

3

^가계

(36.7-45.3%)

^의

3

^배체^처리군에^비하여^낮은^부화율을 보였다

(P<0.05). 2

배체의비정상개체발생률은대조군과전암

컷킹넙치의경우

0-20.4%

^로^사용한^수컷^가계에^따라^큰^차이

를보였다

.

^저온^처리군의^경우에는^대조군

(1.6%)

^과

‘0219’

^가 계

(3.6%)

의

3

배체처리군이

‘1697’

가계

(68.5%)

와

‘1788’

가계

(24.5%)

^의^{처리군보다}^유의하게^낮게^나타나는^것으로^확인되

었으나

, ‘0335’

가계는난황흡수전모두폐사하여유도된전암

컷

3

^배체는^동일^처리조건^임에도^불구하고^생존율이

0-43.5%

로다양하게나타나

,

^사용된^{자성발생성}^수컷^킹넙치의^유전^형 질이생존율에영향을미치는것으로나타났다

.

더욱이난황이 흡수된전암컷

3

^배체^킹넙치

^자어라도

‘1697’

^과

‘1788’

^가계의 경우먹이활동을전혀하지않아부화후

12

일경에는대다수의 개체가폐사하는것으로관찰되었다

.

^그러나

‘0219’

^가계는^대 부분의개체들이종자크기로성장하였다

.

현장에서 빠르고간편한넙치의

3

배체분석방법을확인하 기위해우선

2

^배체와

3

^배체의^세포^및^핵의^크기를^분석한^결 과

(Fig. 1a, 1b),

본방법은경제적이고간편하나

Wolters et al.

(1982)

^의^방법대로

100

^개^정도의^세포^및^핵의^크기를^측정하

여통계처리해야함으로현장에서사용하기에는많은시간을 요하는것으로나타났다

.

Flow cytometry

^에^의한^배수체^분석은^가장^정확하고 ^효율

적인방법으로 대다수의연구실에서이용되고있다

(Aldridge et al., 1990).

^본^{연구에서도}

Fig. 1c

^와^같이

2

^배체와

3

^배체의

구별이뚜렷하게나타나효율적인방법으로인정되었다

.

^그러 나

flow cytometry

분석을위한장비가매우고가임을고려할 때양식어민들이현장에서사용하기에는적당하지않은것으 로보인다

.

유도된배수체에대한염색체분석은그수와핵형을분석하 는방법으로가장정확한분석법으로서이미

Kim et al. (1994a, 1994b)

에의해넙치의

3

배체의염색체는

72

개의염색체로구 성되어있음이보고된바있다

.

^본^{연구에서도}^{일반염색에}^의한 염색체분석결과

,

염색체수가

72

개로나타나

3

배체임이확인 되었다

(Fig. 1d).

^그러나^핵형^분석결과^모두

acrocentric

^염색 체로구성되어있어쉽게

3

^배체를^판단할^수^있는^마커로^사용 할수있는염색체가확인되지않았다

.

이에

NOR

염색체

band- ing

^을^실시하여^그^핵형을^분석한^결과

,

^유도된

3

^배체^킹넙치의 중기염색체상에서가장큰

acrocentric

염색체

3

개에서

NOR band (Fig. 1e)

^가^확인되어

,

^효율적인^배수체^{분석방법으로}^판

명되었다

.

^그러나^어류의^염색체^분석^및

banding

^을^위하여는

전문적기술이필요함을고려할때양식어민들이현장에서사 용하기에는효율적인방법이아닌것으로판단된다

.

지느러미조직의일부를잘라세포를분리한후

silver staining (Goodpasture and Bloom, 1975)

^을^수행한^결과

,

^분석된^핵^내 에서

3

^개의^진한^점

(silver staining positive spot)

^이^{확인되었다}

(Fig. 1f).

이방법은어체를살려두고최소한의조직을얻어염

색까지

60

^분^이내에^분석이^가능하며

,

^세포^및^핵의^크기^측정 방법과달리핵내

3

개의점만을확인하면되므로매우단순하 고신속한방법으로현장에서쉽게

3

^배체^킹넙치를^판별할^수 있는효율적인방법으로확인되었다

.

지느러미 조직의 silver staining 방법

1.

^저온에서^마취시킨^{어체로부터}^적정량의^조직을^얻는다

.

2.

조직을슬라이드위에놓고면도칼로잘게자른다

.

3.

^잘게^자른 ^조직을 ^튜브에^넣고 ^저장액

(0.075 M KCl)

^에

5-10

^분간^처리하여^세포를^{팽창시킨다}

.

4.

상등액을제거한후

,

고정액

(ethanol : glacial acetic acid = 3:1 )

^을^넣고

5

^분간^방치한다

(2-3

^회^반복

).

5.

고정된조직을 핀셋으로 꺼내 슬라이드에 놓고 말린 후

50% acetic acid

^를

1-2

^방울^{떨어뜨리고}^핀셋으로^두들겨 조직을분쇄한다

.

6.

슬라이드를건조시킨후

, 50% AgNO

₃용액과

gelatin solu- tion (2% gelatin in 1% formic acid)

^을^슬라이드^위에

2:1

^의 비율로떨어뜨린후커버글라스를덮고슬라이드가갈색이

되도록실온에서

10-15

^분간^방치한다

(

^온도가^높으면^반응

시간을줄임

).

7.

흐르는물로수세하고건조시킨후현미경으로관찰한다

. 8.

^팽창된^{세포들에서}

3

^개의^진한^점을^확인하여

3

^배체^여부

를확인한다

.

Table 1. Comparisons of hatching rate, incidence of abnormal lar-

vae and survival rate of experimental groups in King-Nupchi strain of olive flounder Paralichthys olivaceus

Experimental

group Hatching rate (%)

Incidence of abnormal larvae

(%)

Survival rate (%) Control 2N 75.7±2.5^a 11.5±1.4^def 67.0±3.2^a

3N 44.2±8.6^bc 1.6±3.1^f 43.5±8.4^b

0219 2N 77.4±11.6â 14.6±7.6^cde 66.6±15.6â 3N 26.0±9.4^d 3.6±7.1êf 24.6±6.5^cd

0335 2N 43.6±14.1^bc 20.4±5.2^cd 34.7±11.7^bc 3N 44.2±12.9^bc 100.0^a 0.0^e

1697 2N 48.7±9.6^b 7.3±7.0^ef 45.6±11.7^b 3N 36.7±7.1^bcd 68.5±14.9^b 12.0±6.7^de

1788 2N 30.4±17.9^cd 0.0^f 30.4±17.9^bc 3N 45.3±9.3^bc 24.5±13.2^c 34.7±12.3^bc Means within a column with different superscripts are significantly different (P<0.05)

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전 암컷 3배체 넙치에 대한 세포유전학 분석법

673 사 사

본 연구는 농림축산식품부

∙

_{해양수산부}

∙

_{농촌진흥청}

∙

_산림청

의지원과제수출용킹넙치배수체종자개발

(

^과제번호

213004-

04-4-SB220)

^에^의해^{수행되었습니다}

.

References

Aldridge FJ, RQ Marston and JV Shireman. 1990. Induced trip- loids and tetraploids in boghead carp, Hypophthalmichthys

nobilis, verified by multi-embryo cytofluoremetric analysis.

Fig. 1. Cytogenetic analysis of triploid KingNupchi strain of olive flounder Paralichthys olivaceus. a, erythrocytes of diploid (nuclear volume, 16.70±0.24 µm³); b, erythrocytes of triploid (nuclear volume, 29.15±2.10 µm³); c, flow cytometry histogram in diploid (red, 1.42 pg/cell) and triploid (black, 2.14±0.03 pg/cell); d, metaphase spread (3N=72); e, NOR-banded metaphase. Arrows indicate NOR-banded chromosomes; f, silver stained nucleus of caudal fin. Bar indicates 10 µm.

100 200 300 400 500 600

N o. o f ce lls

c d

f e

a b

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고민균

ㆍ

정효선

ㆍ

이효빈

ㆍ

김동수

674

Aquaclutre 87, 121-131.

Goodpasture C and Bloom SE. 1975. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53, 37-50.

Jung HS, Kim YK, Kim HC, Noh JK, Lee JH and Kim DS.

2014. Cytogenetic analysis of starry flounder Platichthys

stellatus from Korea. Korean J Fish Aquat Sci 47, 431-434.

http://dx.doi.org/10.5657/KFAS.2014.0431.

Kang KL. 2016. Production of gynogenetic male in KingNupchi strain of olive flounder (Paralichthys olivaceus). MS Thesis, Pukyong National University, Korea.

Kim DS, Moon YB, Jeong CH, Kim BS and Lee YD. 1994a.

Produciton of all-female diploid and triploid populations in

Paralichthys olivaceus. J Aquaculture 7, 159-164.

Kim DS, Jeong CH, Lee YD and Rho S. 1994b. Triploidy induc- tion in olive flounder, Paralichthys olivaceus. J Aquaculture 7, 55-61.

Kligerman AD and Bloom SE. 1977. Rapid chromosome prepa- rations from solid tissues of fishes. J Fish Res Board Can 34, 249-261.

Thorgard GH. 1986. Ploidy manipulation and performance.

Aquaculture 57, 57-64.

Wolters WR, Libey GS and CL Chrisman. 1982. Erythrocyte nuclear measurement of diploid and triploid channel catfish,

Cytogenetic Analysis of All-Female Triploid Olive Flounder Paralichthys olivaceus for Ploidy Verification

671

Copyright © 2016 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

Korean J Fish Aquat Sci 49(5),671-674,2016

Short Communication

(Paralichthys olivaceus)

‘

(KingNupchi)’

, 2013

golden seed project (GSP)

(Jung et al., 2014).

3

90%

3

(Thorgard, 1986).

3

3

.

3

3-4

, Kang (2016)

4

(0219, 0335, 1697

1788)

,

3

2-4℃

45

. 1

30-50 cc

110

3

,

3

.

,

.

18-21℃

.

3

, flow cytometry,

, Ag-NOR banding

solid tissue technique (Klingerman and Bloom, 1977)

silver staining

.

4

,

Table 1

.

2

‘0219’

75%

, ‘0335’, ‘1697’

‘1788’

30.4-48.7%

(P<0.05).

‘0219’

3

26.0%

(44.2%)

고민균·정효선·이효빈·김동수

*

Cytogenetic Analysis of All-Female Triploid Olive Flounder Paralichthys olivaceus for Ploidy Verification

Min Gyun Ko, Hyo Sun Jung, Hyo Bin Lee and Dong Soo Kim 1 *

Department of Fisheries Biology, Pukyong National University, Busan 48513, Korea

Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan 48513, Korea

); flow cytometry (2.14±0.03 pg/cell); chromosome count (3N=72); Ag-NOR banding; and silver staining. Silver staining of finned cells obtained using a solid tissue technique was the most effective method of ploidy verification.

Key words: KingNupchi, Paralichthys olivaceus , Triploid, Silver staining

http://dx.doi.org/10.5657/KFAS.2016.0671 Korean J Fish Aquat Sci 49(5) 671-674, October 2016

Received 9 September 2016; Revised 21 October 2016; Accepted 22 October 2016

*Corresponding author: Tel: +82. 51. 629. 5914 Fax: +82. 51. 629. 5908

E-mail address: [email protected]

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672

3

(36.7-45.3%)

3

(P<0.05). 2

Min Gyun Ko, Hyo Sun Jung, Hyo Bin Lee and Dong Soo Kim ¹ *