Using the genetic resources of common carp for R&D purposes, on the example of the Eurocarp project

Zsigmond JeneyResearch

Institute

for

Fisheries, Aquaculture

and

Irrigation, Szarvas, Hungary

Workshop on Characterization and Conservation of Common Carp Genetic Resources

Szarvas, Hungary, December 4-5, 2007

OUTLINE

•

Introduction

to

Eurocarp Project•

The unique

genetic

resource

of

the

live

gene

bank•

The use

of

the

live

gene

bank in

an

R&D

project

Eurocarp Project

Administrative information

•

Project acronym: EUROCARP•

Project’s official full title: Disease and Stress Resistant Common Carp: Combining Quantitative, Genomic and Proteomic and Immunological marker technologies to identify high performance strains, families and individuals.

•

A projekt

típusa: EU-STREP •

Research priority: 1.3. Modernisation

and sustainability of fisheries,

including aquaculture-based production systems•

Project’s webpage: www.haki.hu/eurocarp

•

Contract no of the project: FP6-2004-SSP-4-022665•

Start date: 1 January 2006

•

Completion date: 31 December 2008

•

STREP: Specific Targeted Research Projects•

SSA: Specific Support Action

Eurocarp consortium

members

Coordinator:

Zsigmond JeneyResearch

Institute

of

Fisheries, Aquaculture

and

Irrigation

Anna Liget 8, Szarvas H-5540, Hungary Tel: +36 66 515 314Fax: +36 66 312 142e-mail: jeneyz@haki.huWebsite: www.haki.hu

PartnersUniversity of Stirling, Institute of AquacultureUnited KingdomBrendan McAndrew

-

b.j.mcandrew@stir.ac.uk

Centre for Environment, Fisheries and Aquaculture ScienceUnited KingdomPeter Dixon -

p.f.dixon@cefas.co.uk

University of Liverpool Laboratory for Environmental Gene Regulation

United KingdomAndrew R Cossins

-

cossins@liv.ac.uk

Akvaforsk

-

The Institute of Aquaculture ResearchNorwayNick Robinson -

nick.robinson@akvaforsk.no

Russian Federal Research Institute of Fisheries and Oceanography

-

Division of Molecular GeneticsRussiaNikolai

Mugue

-

mugue@molgen.vniro.ru

Federal Centre of Fish Genetics and SelectionRussiaAndrey

K Bogeruk

-

fsgcr@ipc.ru

The consortium

Eurocarp Project

Abstract

The project aims at the inclusion of disease and stress resistance as traits within breeding programmes, which will require the use of modern quantitative and molecular genetic tools. Disease resistance has proved to be a difficult traitto assess and improve in fish, direct challenges on potential broodstock

run the risk of turning such fish into carriers.Functional genomics, proteomics and gene mapping will identify candidate genes and QTLs

for resistant fish without achallenge. Heritability estimates for viral and bacterial resistance and genetic correlation to performance traits will beassessed. Differences in gene expression within high and low, viral and bacterial, resistance families with and withoutchallenge will be assessed using a 20K gene carp microarray. Differences in serum/plasma protein expression andimmunological parameters will also be assessed. Congruence between expression levels and protein production willprovide evidence for the importance of particular genes or Gene ontologies

(GO) in these processes. Progress will alsobe made towards development of a second generation medium resolution gene map an important tool for the futureimprovement of carp strains. The results from the quantitative genetic and molecular data will be modelled

to inform onthe optimum design of future practical breeding programmes. This information being disseminated via establishedinternational networks.

“Prehistory”

of

Eurocarp Project

1.

Existance

of the Live Gene Bank of Common Carp

•

HAKI established more than 40 years ago the largest live collection of common carp strains.

•

Dr. János Bakos, the “father of the gene bank”

participates in the maintenance.

•

The basic function of the gene bank has been changed, by shifting towards research on gene bank.

•

In this research activity highest priority is given to stress and disease resistance, as well as to the use of the gene bank in re-habitation of natural waters with preserved strains.

Prehistory

of

Eurocarp Project

2.

Stress

and

disease

resistance

studiesResicarp

Project

Hungarian

national

R&D Project2002-2005

This

project also

based

on

the

live

gene

bank

Amur wild

Tata scaly carp

Duna wild carp

Phenotype of the Szarvas 215 hybrid and its parental lines

Hypoxia tolerancia

Változatok

Túlé

lési

dő,

per

c

-10

0

10

20Sz 22 Sz 215 Sz 15 Tatai Amuri Dunai

22 215 15 Tatai Amuri Dunai

Só tolerancia

Változatok

Túlé

lési

idő,

per

c

-10

0

10

20

Sz 22 Sz 215 Sz 15 Tatai Amuri Dunai

22 215 15 Tatai Amuri Dunai

Cummulative mortality

time, hours

0 20 40 60 80 100 120 140 160

cum

mul

ativ

e m

orta

lity

(CFU

2 x

107.

7 ), %

0

10

20

30

40

50

60

70

80

90

TataDuna22

Állomány változás

300

350

400

450

500

550

600

650

2005

.06.06

2005

.06.07

2005

.06.08

2005

.06.09

2005

.06.10

2005

.06.11

2005

.06.12

2005

.06.13

2005

.06.14

2005

.06.15

2005

.06.16

2005

.06.17

2005

.06.18

2005

.06.19

2005

.06.20

2005

.06.21

2005

.06.22

2005

.06.23

2005

.06.24

2005

.06.25

Dátum

Egye

dszá

m, d

b

TxT 1.AxA 2.DxD 3.22x22 4.15x15 5.215x215 6.22x22A 7.22x22M 8.22xDA 9.22xDM 10.

Prehistory of Eurocarp Project 3. Genetic

studies

Different national projects between 2001 and 2005

•

genetic characterisation of the ex situ gene bank of common carp•

natural and cultured lines/forms/races were investigated

•

15-50 specimens/populations were needed•

investigations based on multiloci microsatellite markers:

•

8 markers tested, 7 of them work well•

MFW1, MFW4, MFW6, MFW7, MFW16, MFW28, MFW31

•

separation by PAGE•

descriptive parameters: e.g., heterozygosity/homozygosity (inbreeding), allele numbers/loci

•

inferring of phylogenetic relationships is also possible based on multiloci data sets

koi outgroup

Amur scaly (Asian wild line)

cultured carp lines kept ingene bank

Danube scalyTisza scaly

(European wild lines)

Relationships

of

carp

lines

using microsatellite markers

- A basal and distinct phylogenetic position of the Asian population has been proved.- Among European populations, “wild” (natural) lines were clearly separated form cultured ones and from each other, too.

- The cultured lines in Hungary showed closer genetic relationship to each other according their known breeding history.- Inbreeding effects at a certain extent could be observed as a result

of the loss of heterozygosity.

•

Criterias

used

for

selecting

lines–

known

origin

and/or

documented

breeding

history

–

preliminary

data

available

about

genetic

and

immunological parameters: more detailed

data

are

available

from

previous

studies

(e.g., Resicarp)–

sufficiently

different

genetic

background

(„gene

pool”) and/or

immunological

potential–

four

lines

should

be used: two

natural

and

two

cultured

lines

are

recommended

Carp

lines

for

EUROCARP

1

Amur scalyDanube scalySzarvas 15 mirrorSzarvas 22 mirror

Szeged mirror

Tata mirrorTata scaly

Amur scaly

•

The Amur wild

carp

is an

„ancient”

form

that

originated

from

the Asian

carp

centre (Amur-China

type

of

wild

carp, Cyprinius carpio

haematopterus).

•

During

the

centuries, after

settling

in

the

river

Amur, this

carp

has been

adapted

to

the

local

environmental

conditions.

•

It

was

brought

into

the

gene

bank in

Szarvas in

1982 from

the Russian

National

Fisheries

Research

Institute.

•

26 specimens

are

currently

in

gene

bank (+ ca. 20 specimens

in Resicarp population).

Danube

scaly

•

The Danube

scaly

carp

is a „wild”

form

that

originated

from

the Danube

river

and

its

tributaries.

•

It

was

introduced

to

the

gene

bank in

Szarvas in

the

early

1990s.

•

36 specimens

are

currently

in

gene

bank (+ ca. 20 specimens

in Resicarp population).

Tata scaly

•

The Tata scaly

carp

is one

of

the

oldest

forms

of

cultivated

common carp

in

Hungary. The first

documentation

available

about

its

introduction

from

Germany

dates

back

to

1860, but

from

1890 there are

references

to

the

import of

scaly

carps

from

Trebon

(Czech

Republic) to

Tata (Antalfi

1971).

•

The rapid growth

rate

and

the

round

body

shape

has been

preferred during

the

conscious

selection

activity. As

a scaly

population, it

has

contributed

to

the

development

of

several

scaly

Hungarian

common carp

strains

at

the

beginning

of

the

20. century.

•

21 specimens

are

currently

in

gene

bank (+ ca. 20 specimens

in Resicarp population).

Szarvas 15

mirror

•

The carp

line

Szarvas 15

was

derived

by

individual

selection

from the

Biharugra

and Hortobágy

strains. After 1964 it was used in

many crossing experiments e.g.

males of the Szarvas mirror carp are used in production of the Szarvas 215

•

Quantitative

traits, such as fast growth and high fecundity

were used

as

selection

criteria.

•

As a family founder male (two line hybrids 15) produced outstanding hybrid combinations such as the state approved Szarvas 215

•

45

specimens

are

currently

in

gene

bank (+ ca. 20 specimens

in Resicarp population).

Eurocarp Project: Activities

1.-18. monthIn order to implement the project activities, 96 common carp families have been established during the1st year of the

project.

Four genetically distinct common carp strains/varieties (Duna

scaly, Amur

scaly, Tata

scaly andSzarvas 15, mirror)

have been used for a specially designed cross-scheme.

Progenies (2006 generation) created were grown in a closed

system of HAKI, Szarvas, according to a modified

technology of carp nursing

and fingerling growing.

Separate culture

units were applied, until the „tagging size”

was reached.

During August and

early September 9,600 carp (100 of eachfamily) has been tagged and grouped for5 different

purposes: –

progeny tests in

pond conditions–

bacterial

(Aeromonas hydrophila) infection–

viral (Koi Herpes Virus) infections –

reserve

groups–

stresss

tudies

Eurocarp Project: Activities

1.-18. monthProgeny

tests in

pond conditions: harvested

in

October

2007, results

are

being anaylsed

Disease

challenges

in

2 rounds:1.round: Artificial

infection

of

96 families, mortality

and

production

data

analysed

=>

10 high

and

10 low

families

determined2. round: 10 high

and

10 low

families

are

challenged

again

=>

sampling

for

genomic, proteomic

and

immunological

studiesDisease

challenge was

carried

out with

bacterial

(Aeromonas hydrophila) and

viral pathogens

(Koi Herpes Virus)

Genomic, proteomic

and

immunological

studies

are

in

progress

In

May

2007, nineteen

families

(including

„high

and

low

bacteria”

as

well

as

„high

andlow

virus”

varieties

have

been

produced

again

(2007 generation). Their

testing(production

traits,stress

resistance

traits, etc) is in

progress.

In

August

2007 (half lifetime of the project) we have produced our 18-month ProgressReport.

Outline structure of

the

project

BREEDING, REARING AND TAGGING OF FISH

DIALLEL CROSSES

MAPPING CROSSES

MODELLING OF SELECTION

DEVELOPMENT AND SCREENING OF MARKERS IN

PARENTS

LINKAGE MAP INCLUDING

DISEASE/STRESS EST MARKERS

HIGH AND LOW FAMILIES

COMMERCIAL GROWOUT

QUANTITATIVE GENETICS

PARAMETERS

BACTERIAL CHALLENGE

VIRAL CHALLENGE

ANALYSIS

DISEASE/STRESS MICROARRAY, SCREENING OF HIGH AND LOW

FAMILIES

PROTEOMICS, IMMUNE

PARAMETERS

VIRAL CHALLENGE

BACTERIAL CHALLENGE

BREEDING, REARING AND TAGGING OF FISH

DIALLEL CROSSES

MAPPING CROSSES

MODELLING OF SELECTION

DEVELOPMENT AND SCREENING OF MARKERS IN

PARENTS

LINKAGE MAP INCLUDING

DISEASE/STRESS EST MARKERS

HIGH AND LOW FAMILIES

COMMERCIAL GROWOUT

QUANTITATIVE GENETICS

PARAMETERS

BACTERIAL CHALLENGE

VIRAL CHALLENGE

ANALYSIS

DISEASE/STRESS MICROARRAY, SCREENING OF HIGH AND LOW

FAMILIES

PROTEOMICS, IMMUNE

PARAMETERS

VIRAL CHALLENGE

BACTERIAL CHALLENGE

Progress of

the

project

Infrastructure development: rearing units for large number of families

Before After

Infrastructure

development: experimental unit for infection studies

Broodstock maintenance, selection

of

breeders

Planning, artificial reproduction

Crossing schemes

Duna

Amur

Tata

15

D1xD1 A1xD7 T1xD3 15-1xD9 D1xD2 A1xD8 T1xD4 15-1xD10 D2xD3 A2xD9 T2xD5 15-2xD1 D2xD4 A2xD10 T2xD6 15-2xD2 D3xD5 A3xD1 T3xD7 15-3xD3

Duna

D3xD6 A3xD2 T3xD8 15-3xD4

D1xA5 A1xA1 T1xA7 15-1xA3 D1xA6 A2xA2 T1xA8 15-1xA4 D4xA1 A4xA7 T4xA3 15-4xA9 D4xA2 A4xA8 T4xA4 15-4xA10 D5xA3 A5xA9 T5xA5 15-5xA1

Amur

D5xA4 A5xA10 T5A6 15-5xA2

D2xT1 A2xT7 T2xT3 15-2xT9 D2xT2 A2xT8 T2xT4 15-2xT10 D3xT3 A3xT9 T3xT5 15-3xT1 D3xT4 A3xT10 T3xT6 15-3xT2 D4xT5 A4xT1 T4xT7 15-4xT3

Tata

D4xT6 A4xT2 T4xT8 15-4xT4

D1x15-3 A1x 15-9 T1x15-7 15-1x15-1 D1x15-4 A1x15-10 T1x15-8 15-1x15-2 D2x15-5 A2x15-1 T2x15-9 15-2x15-3 D2x15-6 A2x15-2 T2x15-10 15-2x15-4 D5x15-1 A5x15-7 T5x15-3 15-5x15-9

15

D5x15-2 A5x15-8 T5x15-4 15-5x15-10

male

Duna

Amur

Tata

15

No 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

1 X X X 2 X X X 3 X X X 4 X X X 5 X X 6 X X 7 X X 8 X X 9 X X

a

10 X X

1 X X X 2 X X X 3 X X X 4 X X X 5 X X 6 X X 7 X X 8 X X 9 X X

ur

10 X X

1 X X X 2 X X X 3 X X X 4 X X X 5 X X 6 X X 7 X X 8 X X 9 X X

a

10 X X

1 X X X 2 X X X 3 X X X 4 X X X 5 X 6 X 7 X X 8 X X 9 X X X 10 X X X

D A T 15

D

A

T

15

Artificial

reproduction

Rearing technology of carp fingerlingPropagation•

The broodstock

originated from Resicarp Project.

•

Hormonal induced spawning (pre dose: female: 0.5mg carp hypophysis/kg, male: 1mg CH/kg, main dose: female: 4mg CH/kg, male -)

•

The fertilized eggs were sticked

to artificial nests (eggs of 1 line/crossing on 2 nests)

•

Hatching in small tanks (40L) separately

Rearing•

After hatching the number of fish larva is reduced to 2000/tank

•

Feeding: Artemia

nauplii

in the first 2 weeks (5 nauplii/ml, 5 times a day).•

Weaning in the 3rd week

•

From the 4th week feeding with artificial carp diet only (Nutreco)•

Restocking (1000 larvae) into the large rearing tanks (300L)

•

Continuous control and reduction of the number of fingerlings: the final density is 200 fingerlings/tank, at the marking size (5-8 g).

•

The fish health status is supervised by veterinary service

Incubation, larval

and

fingerling

rearing

Transportation

of

carp

to Weymouth, UK

Gene mapping: Sampling

for VNIRO, with simultaneous sexing the fish

See

crossings

mintavételek mapping

060926

Individual

tagging

The HAKI-team

Why Eurocarp Project succeeded?

•

Factors at national level

–

Values of common interest (Carp gene

bank)–

Experience in running complex projects at national level

–

Working

relationship

with

national

R&D institutions in Hungary

–

Measurable scientific results/achievements–

Institutional background

Why Eurocarp Project succeded?

•

Factors at international level

–

Experience in participating/running complex projects at international level

–

Working

relationship

with

international

R&D institutions in Europe

–

Lobbying–

LUCK!

–

Others (please do not ask☺)

Thank You for your attention! Köszönöm a figyelmet!

FVM*/HAKI-CGIAR/WFC Workshop on Carp Genetics

HAKI, Szarvas, HungaryDecember 4-6, 2007

Major objectives

of

the

workshop:

•

Review

the

state

of

art of

R&D activities

in

carp

genetics

generally and

in

the

context

of

European-Asian

collaboration.

•

Explore

the

possibility

of

collaboration

between

major institutions involved

in

carp

genetics

taking

into

previous

INGA experiences.

•

Elaborate

a proposal

for

possible

follow

up

of

INGA activities

in

the field

of

carp

genetics

and

breeding.

* - FVM: Ministry

of

Agriculture

and

Rural

Development-

CGIAR: Consultative

Group

for International Agricultural Research-

WFC: Worldfish

Center