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HELLENIC CENTRE FOR MARINE RESEARCH, Institute of Marine Biology and Genetics, Crete

Recent developments in Recent developments in

sea bass and seabream sea bass and seabream

mappingmapping

Elena Sarropoulou

Paris 7-8th February, 2008


Hellenic Centre for Marine ResearchHellenic Centre for Marine Research

Institute of Marine Biology and GeneticsInstitute of Marine Biology and Genetics

•Institute of Oceanography

•Institute of Aquaculture

•Institute of Marine Biological Resources

•Institute of Marine Biology and Genetics

•Institute of Inland Waters


Institute of Marine Biology and Genetics Institute of Marine Biology and Genetics

CRETE (IMBG)CRETE (IMBG)

“THALASSOKOSOMOS: IMBG AND CRETAQARIUM“THALASSOKOSOMOS: IMBG AND CRETAQARIUM

Director: Dr. A. Magoulas Director: Dr.P.Divanach


Recent developments in Recent developments in

sea bass and seabream sea bass and seabream

mappingmapping


Biological MotivationBiological Motivation

� Detection of molecular markers linked to phenotypes which

are of importance for aquaculture such as growth, body shape,

color and disease resistances.

�Detection of candidate genes responsible for quantitative and

qualitative traits.

� Detection of syntenic relationships and conserved regions

among teleosts.

� Insights of the genomic make up of sea bream and sea bass


Zebrafish tumors caused by

mutation of a ribosomal

protein gene

Seabream/ Sea bass:

variations between

individuals (which are

inherited to the next

generation).

•Growth

•Shape

•Disease resistance

•Skeletal dimorphism

•Color mutations


Molecular toolsMolecular tools

• Molecular markers

=> microsatellite markers, EST-SSRs

=>SNPs

• Maps

=>Genetic linkage map

=>Radiation hybrid map

=>Physical map

• QTL mapping


Microsatellite markers(Also known as –Tandem simple sequence repeats (SSRs) or Variable

Number of Tandem Repeats (VNTRs) or short tandem repeats (STR) or

simply Microsatellites)

Tandemly repeated sequences whose unit of repetition is between one and six bp. Most commonly used repeats GA or CA

and GATA and GACA

Classified in three families:

– Pure: CACACACACACACACACACA

– Compound: CACACACACAGAGAGAGAGA

– Interrupted: CACATTCACACATTCATTCA


TWO ALLELES

• Heterozygous

CGTAGTCATCACACACACACACACACGTAGCG

CGTAGTCATCACACACACACACACACACGTAGCG


Molecular toolsMolecular tools

• Molecular markers

=> microsatellite markers, EST-SSRs

=>SNPs

• Maps

=>Genetic linkage map

=>Radiation hybrid map

=>Physical map

• QTL mapping


Single Nucleotide Polymorphism


Candidate gene approach:

combining phenotypic analysis of the genes function with

information about the approximate genetic map location. Cloning by

the candidate gene approach begins with an in depth analysis of

phenotype in order to define the genes’ biological function (e.g for

mutations which result in fish that lack all pigment, the gene is likely

to code for an enzyme involved in melanin biosynthesis) .

Positional cloning or recombinant mapping:

relies on the use of polymorphisms that occur between the mutant strain and

the mapping strain, where the mapping strain is crossed with the mutant

carrier to generate a mapping line from which mutant and wild type sibling

embryos are collected. The identification of polymorphic markers that are

closely linked to the mutation, through comparisons of wild type and mutant

embryos, is a critical step in any positional cloning project.


Three different kinds of MAPs

• Genetic Map

• Radiation hybrid map

• Physical map

• Genetic Map


• Physical map


Genetic linkage map

12.1 %

12.1 %


Second generation linkage map and synteny analysis of European sea bass

Chistiakov D., J. Lagnel, C. S. Tsigenopoulos, C. Haley, B. Hellemans, F.A.M. Volckaert, G. Kotoulas

Department of Pathology, University of Pittsburgh Medical Center, A719 Scaife Hall, 3550 Terrace Street,

Pittsburgh, PA 15261, USA

Hellenic Centre for Marine Research (HCMR) Crete, Institute of Marine Biology and Genetics (IMBG), T

halassocosmos, Institute of Marine Biology and Genetics, P.O.Box 2214,, 715 00 Heraklion, Crete, Greece

Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Charles Deberiotstraat 32, B-3000 Leuven, Belgium

Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UKScotland

submitted


Second-generation linkage Map (in prep)

– 196 microsatellites, 176 AFLPs and 2 SNPs

– 36 linkage groups / sex-averaged map spanned 1373.1

cM

– Sixty seven markers (11 microsatellites, 47 AFLPs and

9 SNPs) remained unlinked

– The current linkage map includes 24 anonymous type I

microsatellites derived from ESTs and 6 genes

including peptide Y, CYP19, SOX10, PXN1, ERA and

TCRB


Genetics 174: 851–861 (October 2006)

First Map: (Franch et al, 2006)

204 markers (7 EST-SSRs and 197 SSRs)

Unlinked:6, un-positioned: 7

26 linkage groups, 1241.9 cM

SEABREAMSEABREAM



Second-generation linkage Map (in prep)

– 196 microsatellites, 176 AFLPs and 2 SNPs

– 36 linkage groups / sex-averaged map spanned 1373.1

cM

– Sixty seven markers (11 microsatellites, 47 AFLPs and

9 SNPs) remained unlinked

– The current linkage map includes 24 anonymous type I

microsatellites derived from ESTs and 6 genes

including peptide Y, CYP19, SOX10, PXN1, ERA

and TCRB

SEABASS:


Second MapSecond Map (Aquafirst, in prep)

–327 markers (105 EST-SSRs and 222SSRs)

–Unlinked: 16, un-positioned: 4

–26 (to 29) linkage groups, 2221 cM

SEABREAM:


Three different kinds of MAPs

• Genetic Map


• Physical map

• Genetic Map


• Physical map


Seabream Hamster

Each clone retain ~30 % of the seabream genome. Linkage is determined by statistical

analysis of co-retention frequency.

Radiation

Senger et al. 2006

Radiation hybrid mapping: Method to map ESTs, genes Radiation hybrid mapping: Method to map ESTs, genes

and other DNA’sand other DNA’s


PipelineTemplate DNA dilution in 96 deep well plates

Oligos 20µΜ in 96 Cluster tubes

PCR


Genotyping


cDN07P0006M12 200000002000000000000000000000000000000000000000000000000000000000000000000000000000000000000

SA-E10A_H05 200000002000000000000000000000000000000000000000000000000000000000000000000000000000000000000

SA-E11C_A11 200000002000000000000000000000000000000000000000000000000000000000000000000000000000000000000

cDN08P0005J15 200000002000000000000000000000000000000000000000000000000000000000000000010001000000000100000

cDN07P0002O 22 200000002000000000000000000000000000000000000000222002002022020100012200012122000000010100000

SA-E10C_H1 200000002000000000000000000000000000000000000010100000010100001000000000000000001000000100011

cDN04P0001J09 200000002000000000000000000000000000000000001000000000000000000000000100000011000000011101100

cDN12P0006G1 0 200000002000000000000000000000000000000000100000000000000000000000002000000020000000000000000

cDN04P0005B05 200000002000000000000000000000000000000010000000010001000110000000000000000001000000000000011

cDN04P0003N2 2 200000002000000000000000000000000000000100000000000000000100000000010000000000000000000000000

cDN11P0004L03 200000002000000000000000000000000000100000100000000000000001000100012011001100010000010100011

cDN11P0001A02 200000002000000000000000000000000001000000001000010100010000000110000001000100000000000000101

cDN02P0003C11 200000002000000000000000000000001000000100000000010000010000000000000000100000000100100100101

cDN03P0005L10 200000002000000000000000000000001000001000000010110000000110000001000000000001001000000000000

cDN01P0006B15 200000002000000000000000000000002000000000000101000000000110000000000000000001001000000000000

cDN02P0003M14 200000002000000000000000000000010000000000000000000000000000000000000000000000000000000000000

cDN08P0005J24 200000002000000000000000000000100100000000100000001000000101100000000100000000111011111000000

SAPD01676|TET2A 200000002000000000000000000100000000000000000000000000000000000000000000000000000000000100200

cDN03P0006E09 200000002000000000000000000100000001000000001000000000000000000000000000000000000000000000000

cDN10P0001F24 200000002000000000000000010001000011000000000001000010001000000000001000000100101000001010000

SAPD02114 200000002000000000000000010100000000000000000000000000000000000000000000000100000000000100000

cDN01P0004L04 200000002000000000000000011000000000100101100010010000000000000000110000000000000000010000000

cDN02P0005N02 200000002000000000000001000000000000001000000000000001000000001000000100000022000000000010000



� 288 microsatellite marker

� 82 genes

� 70 STS


� 428 gene-based markers

� 74 microsatellite marker


Coding sequences for RH

mapping

24%

5%

7%

7%7%3%

19%

28%

Pthrp stimul. Subtract.Larvae head

Kidney

Intervert. Cartilage

Pituitary

Estrog. Stimul. Subtrac.Gonads

Whole body

Liver

Larvae


Type,Number, and Reference of Markers

Typed on the RH panel

D.M.Power

L.Bargelloni

G.Kotoulas

560Type II:

Microsatellites

1400Total

D.M.Power

L.Bargelloni

G.Kotoulas

840TYPE I :

Coding sequences

OriginNumber of

markers on the

actual map

Successfully PCR

amplified

Numbers of

markers

genotyped

Markers

694

463

1157 945

448

497

Senger et al., 2006 and Sarropoulou et al.,

2007


Genetic linkage map vs. RH map


RH group 20Genetic linkage group 1


Comparative analysis

“one of the hopes is that through comparative

analysis knowledge about the genetic make-up of

non-model organisms can be gained without having

to construct a physical map”

K.C.Stemshorn, Q.W. Nolte & D.Tautz, J.Evol.Biol. 2005


An Aquaculture motivated Genetics & Comparative

Genomics project on the gilthead sea bream

Euteleostei

Ostariophysi

Atherinomorpha

Percomorpha

Tetraodontiformes

Cypriniformes

Siluriformes

Beloniformes

PerciformesSparus aurata

Gilthead sea bream

Danio rerio

zebrafish

Ictalurus punctatus

Channel catfish

Oryzias latipes

Medaka

Takifugu rubripes (Fugu)

Tetraodon nigroviridis

Modified after Wittbrodt et al., 2001


Genome data

Sparus

aurata

Danio rerio Fugu

rubripes

Tetraodon

nigroviridis

Genome

(bp)

~1x109

~1,7x109

~4x108

~3,4x108

chromosomes

(n)

N=24

N=25

N=22

N=21


Number of BLAST and BLAT matches of

Seabream sequences (n=794) mapped on the

RH map against Tetraodon, Fugu and Danio

Matches with BLAST search

e<10-4 , >50bp

BLAT search

score>80

Tetraodon 344 (43%) 301 (38%)

Fugu 478 (60%) 378 (47%)

Danio 243 (30%) 90 (11%)


Latin name: Tetraodon nigroviridis

Common name: Pufferfish Order: Tetraodontiformes

Chromosome number: 21

Tetraodon nigroviridis belongs to the family of "smooth" pufferfish (Tetraodontidae)

and, at a higher systematic level, to the order Tetraodontiformes, which also includes

the diodons or "spiny" pufferfish (Diodontidae), sunfishes (Molidae), boxfishes

(Ostraciidae) and triggerfishes (Balistidae), among others. Tetraodon nigroviridis is

often confused with a closely-related species, Tetraodon fluviatilis.

Little fish (less than 10 centimeters

long in captivity) which is popular

with tropical fish fanciers.

In its natural state, it is found in

rivers and streams of Southeast

Asia (Indonesia, Indochina,

Malaysia, the Philippines), as well as

in estuaries and mangrove swamps,

and even occasionally in the sea; it is

therefore not strictly limited to

fresh water.


Comparative mapping to Tetraodon


215Chr8

258Chr7

103Chr6

223Chr5

147Chr4

234Chr3

398Chr2

391Chr1

327Chr11

309Chr12

280Chr10

288Chr9

Marine GenomicsMarine GenomicsTetraodonTetraodon

34Chr20

93Chr19

198Chr18

202Chr17

156Chr16

247Chr15

224Chr14

256Chr13

2148Un_random

6806total

4761subtotal

178Chr21

Marine GenomicsMarine GenomicsTetraodonTetraodon

BLAT search of 31,705 EST sequences generated by Marine Genomics


Comparison of

gene order between

Sparus aurata and

Tetraodon

nigroviridis


Syntenic relationship of

RH group 18 with

Tetraodon chromosome 05

GM201

UNH 995 and

Sparus aurata

gonadal P450

aromatase

Sex determining Sex determining

region in tilapiaregion in tilapia

-- the homologue to linkage

group 1 in Tilapia

(Lee et al., 2003 and 2004)


Syntenic relationship of

RH group 18 with

Tetraodon chromosome 05

GM201

UNH 995 and

Sparus aurata

gonadal P450

aromatase

Sex determining Sex determining

region in tilapiaregion in tilapia

-- the homologue to linkage

group 1 in Tilapia

(Lee et al., 2003 and 2004)


Model fish speciesModel fish species

Three-spined Stickleback

Medaka

Species of Aquaculture interestSpecies of Aquaculture interest

Tilapia

Seabream

Sea bass


Sparus aurata

Gasteroceus aculeatus

Astatotilapia burtoni

Oryzias latipes

Fundulus heteroclitus

Tetraodon nigroviridis

Takifugu rubripes

Danio rerio

Cyprinus carpio

Ictalurus punctatus

Oncorhynchus mykiss

Salmo salar

Homo sapiens

1 0 0

1 0 0

1 0 0

1 0 0

1 0 0

7 2

8 0

6 8

4 4

7 7

0 . 0 2


The three spine stickleback Gasterosteus

aculeatus is an important model in

evolutionary and ethologic studies. Its

utility would greatly be increased by the

availability of molecular markers

distinguishing individuals and populations.

Latin name: Gasterosteus aculeatus

Common name: three spine stickleback Order:Gasterosteiformes


It is a holarctic species of wide distribution

along the Pacific coast of North America,

from Bering Strait, Alaska to Arroyo El

Rosario, Baja California, Mexico, inhabiting

coastal marine waters, brackish waters and a

wide array of freshwater habitats.


Latin name: Oryzias latipes

Common name: Medaka Order: Beloniformes


Medaka is a small (3–4 cm) egg-laying freshwater fish; its eggs are

fertilized and develop externally. Both the eggs and the embryos are

transparent. Medaka is very hardy and tolerates a wide range of salinities

and temperatures (10–40 °C); it is easy to breed and highly resistant to

common fish diseases.

Medaka (Oryzias latipes) has come to

be widely used as a laboratory animal

in various fields in biology, mainly in

developmental biology and genetics.

Its relatively short life cycle, capacity

to reproduce, and ease of breeding

are responsible for its popularity in

these fields.


Marine Biotechnology , in press



Comparative

Genomics via the

genome of model

fish species like

the three spine

stickleback and

tilapia


D. Labrax BAC end sequencingBAC end coverage of T. negroviridis genome

50

Size of Tetraodon Chromosomes(Mb)

0 5 10 15 20 25

Nu

mb

er

of

BA

C e

nd

s

hitin

g c

hro

mo

so

me

0

200

400

600

800

1000

1200

1400

1600

1800

chr01

chr01R

chr02

chr02R

chr03

chr03R

chr04

chr05

chr06

chr07chr08chr09

chr10

chr11

chr12chr13

chr14

chr15

chr15R

chr16

chr17

chr18

chr19

chr20

chr21

chr21R

Y=84.4X

H Khul et al 25% end sequences mapped to Tetraodon genome (blastn e-5)


CTC/AAG-323CMTA170aMC202MC54OPP07-1.07OPP19-1.23

MC296

MG19CTA/AGC-105

MC127MC235

MC221MC209BMC148OPX11-1.01

MC53OPP08-0.64MC244OPY16-0.49OPK03-0.67CTA/AGG-186CAT/AAC-62OPO15-1.78MC331BCM11CM88AMG57MC27CMGA128

MC124

MC298

MC215

MC207B

0

23

20

29

40

44

54

57

60

71

79

85

91

95

AEST90B

CTA/ACGCTA/ACG--302302

MC365MC32

50

CMGA36BCMGA36B

MC373MC373

MC311

Npr1MC44

ACTG4-0.60CTC/AAG-136

CTA/AGG-200CAC/AAC-78CTA/AAC-62CAC/AAC-79

OPJ18-1.24

OPX11-0.59MC253Acs2CACG4-1.23CSAT425BCAC/AAC-119CAC/AAC-77

MC317

CM139

CMGA15

MC387OPG04-1.54MC217MC125

MC249

CAC/ACGCAC/ACG--139139

0

6

26

47

42

51

74

97

102

112

28

61

CMGA21

55

66

106

CTA/AGG-256CAC/AAC-52MC63MC259AEST239BMC255AMC234

MC146

Mlf 1

MC326CAC/AAG-264

MC388

OPL04-0.75Icl1MG23Mas1MC264

CAA/AGC-74CAT/AAG-66

MC375

MC331AMC277OPK03-2.02

MC20MC231

MC40MC118

0

24

16

30

35

55

58

69

76

83

95

101

CAT/AAC-150

MC16MC82

CAT/AACCAT/AAC--5555

MC291Acs1MC349CMGA104MG34BMC93

MC278MC265

CAC/AAGCAC/AAG--130130

5

12

40

47

OPR12OPR12--0.750.75

MC301

MC319MC68CAC/ACC-86

Aox2

CMTC13

MC356CM173

MC11A

MC11B

MC316MC78

Mdr1

CMACC146

MC208

CMAG59

CMAT141

AEST135A

MC138

AEST59

CAC/AAGCAC/AAG--5858

0

6

16

20

33

38

44

54

58

65

73

79

87

93

Aco-2

29

MC287MC233MC7

GATA4-0.71

MC33MC33

CAC/AACCAC/AAC--217217

CAG/AAC-236

CCT/AAC-173

MC4MC223OPM11-1.52CAT/AAC-141OPL04-1.30

CCT/AAC-176MC261A

MC256

CMAT35

GACA4-0.52

CAA/AGG-129

MC76

59

54

41

32

17

10

0

MC99B

36

22

CMTC928

MC247

CAA/AGG-98

OPW08OPW08--1.831.83

MC279

AEST144

CAA/AGC-214

CTC/AAG-68Msf 1

CTC/AAC-71CTC/AAG-115MC120

MG1

MC133B

AEST23AEST1BCAT/AAC-92MC85CM101A

CMCT505CAT/AAG-132CMCAA145OPQ17-1.25MC309MC210OPU01-1.36

0

23

17

36

46

53

61

72

78

94

MC134

MC216MC216

49

67 MC22A

Linkage g

roups N

OT ch

rom

osom

es!


Ordering a linkage group to a

chromosomeFISH technology


1 2 4 6 7 stages

1 2 4 6 7 stages

R2=

0.69

1

A

:

B

:

R2

=

0.6

4

Cluster 3Cluster 2

1 2 4 6 7 stages

1 2 4 6 7 stages

R2

=0.

68

2

R2

=0.

56

8

1 2 4 6 7 stages

- 2.0

- 1.0

0.0

1.0

2.0

3.0

4.0

5.0 cardiac troponin T isomere: AY005139.1

Muscle type creatine kinase AY034097.1

myosin heavy : AB039672.1

myosin light chain: AF150904.1 2

parvalbumin:AY035586.1

fast skeletal muscle troponin T: AF500272

tropomyosin: AB045645

troponin 1: NM_017185.1


OUTLOOKOUTLOOK

Immune

related genes

detection of Quantitative trait loci

marker assisted breeding for quantitative traits

1 2 4 6 7 stages

1 2 4 6 7 stages

detection of functional units


•Sparus aurata prolactin receptor

•Sparus aurata growth hormone receptor

•osteoclast-stimulating factor

•Sparus aurata growth hormone gene

•Sparus aurata prolactin (PRL)

•Sparus aurata osteocalcin gene,


THANK YOU!

Documents

Recent developments in sea bass and seabream mappinggenomics.aquaculture-europe.org › fileadmin › Aquafunc › doc › Aqua… · Recent developments in sea bass and seabream