Genomics tools for aquaculture: Status and perspectives

Luca Bargelloni

Department of Public Health, Comparative Patology, and Veterinary Hygiene

University of Padova

Italy

A variety of species

Inoue et al. 2005

sturgeons

eels

carp

Atlantic salmontrout

Atlantic cod, sea bass, sea bream, turbot, sole, tilapia, tuna

A variety of tools/methodologies

Genetic linkage maps

Physical maps

RH/Happy maps

Whole-genome sequencing

Transcriptomics (mRNA, miRNA)

Epigenomics (DNA methylation)

Proteomics

Metabolomics

…

…

Ron and Weller 2007

QTL detection

Properties and functions that arise from the interacting parts in a system "emergent properties“.

A system is said to be complex if its emergent properties are unpredictable.

Monitoring, predicting, modifying the effects of environmental conditions (sensu lato) on farmed animals

Towards a systems biology approach in aquaculture species?

Ron and Weller 2007

QTL detection

Cyprinus carpio (common carp) >100 yes Sun and Liang 2004, Yue et al 2004 and reference therein

Dicentrarchus labrax (European sea bass)

>374 (μ+AFLPs)

yes Chistiakov et al. 2005, Volckaert personal communication

Gadus morhua (Atlantic cod) >70 no? Westgaard et al. 2007 and references therein

Oncorhynchus mykiss (rainbow trout)

>900 yes Johnson et al. 2007, Guyomard et al. 2006

Scophthalmus maximus (turbot) >350 yes Pardo et al. 2007, Martinez personal communication

Salmo salar (Atlantic salmon) ? yes Moen et al. 2004, Gilbey et al. 2004 ??

Salmo trutta (brown trout) >50 no Lerceteau-Köhler and Weiss (2006)

Solea senegalensis (Senegal sole) 50 End 2007 Castro et al. 2006 , Cerda personal comm.

Sparus aurata (gilthead sea bream)

>320 yes Franch et al. 2006, Kotoulas personal communication

Acipenser spp. (sturgeons) <50 no Congiu personal communication

Crassostrea gigas (Pacific oyster) >100 yes Hubert and Hedgecock 2004

Mytilus edulis (blue mussel) 791 AFLP+μ

yes Lallias et al 2007, Lapegue personal communication

Ostrea edulis (European oyster) >20 yes Lapegue personal communication

Ruditapes philippinarum (Manila clam)

>20 no Saavedra personal communication

Genetic linkage maps

Genetic linkage maps

Prevalently type II (non-coding) markers microsatellites + AFLPs

Comparability of linkage maps

Toward type I (coding) markers:EST-linked microsatellites (STR present in 3-10% fish ESTs)SNPs in coding regions

62/204 (30%) of loci contain conserved sequence regions compared to the pufferfish genomeFranch et al. 2006

Ron and Weller 2007

• Radiation hybrid mapping• Happy mapping

Sea bream primary fibroblasts were γ-irradiated at 3000 rad and subsequently fused with hypoxanthine–guanine phosphoribosyltransferase-deficient (HPRT−) hamster cells (CHO)

Senger et al. 2006

HPRT−

Each clone was stopped at the stage of confluence of 2 × 75-cm2 flasks. At this stage, cells from one flask were frozen and DNA was extracted from the cells in the duplicate flask to perform whole genome amplification (WGA) with Φ29 polymerase

PCR-amplify each marker (no need for polymorphism) on the RH panel

Radiation hybrid map of Sparus aurata consisting 25 radiation hybrid groups and 937 molecular markers.

Sarropoulou et al. 2007 BMC Genomics 8:44

141 more markers addedKotoulas personal comm.

F.Galibert 30/9/07

Sea bass RH panel

* Suitable sequences must exist for use as markers. These can be any stretches of sequence longer than one or two hundred bases. In order to be useful, they need to be unique within the genome. (A multicopy sequence will not give useful mapping data in general.)

* It must be possible to obtain living primary cells from the species to be mapped.

* It must be possible to complement the mutant rodent cells with the genome from the species to be mapped.

* The resolution of the map (its ability to correctly order closely-spaced markers) is proportional to the radiation dose. High doses might not be achievable in all species.

* The maximum range of a RHmap is up to tens of Mb – RH maps are useful to construct coarse-medium density genome maps.

Happy (HAPloid DNA samples using the PolYmerase chain reaction) mapping

break this DNA into random fragments, using either radiation or mechanical shearing

Dilute these fragments down, and dispense a ‘panel’ of very small samples into the wells of a microtitre plate. The samples are so small that each well contains less than a complete set of fragments

Using very sensitive PCR-based methods, we test to see which of the markers is present in each of the samples (‘typing’)

When we analyse the data, in this case, we find that the red and yellow markers often occur together (‘co-segregate’) in the mapping panel (as indicated by the dotted circles). This tells us that they lie next to each other in the genome, whilst the blue marker (which occurs independently) must lie further away. More detailed statistical analysis allows us to calculate the exact locations of the markers in the genome.

* Suitable sequences must exist for use as markers. These can be any stretches of sequence longer than one or two hundred bases. In order to be useful, they need to be unique within the genome. (A multicopy sequence will not give useful mapping data in general.)

* It must be possible to obtain good-quality genomic DNA from the species to be mapped.

* The resolution of the map (its ability to correctly order closely-spaced markers) can be as high as you like - it’s just a matter of making the mapping panel from small enough DNA fragments. However, the range of the panel (the maximum measurable distance between markers) will be about 10 times the resolution. For example, a mapping panel can be made with a resolution of 10kb and a range of 100kb, meaning that markers as close together as 10kb can be reliably ordered, but markers more than 100kb apart will remain un-connected.

•The maximum range of a HAPPY map (the maximum measurable distance between markers) is about one megabase - this limit is set by the size of DNA fragments that can easily be purified. Since the location of the markers isn’t known to begin with, this means that you need at least 1 marker per 100kb of genome on average, to ensure that there aren’t any inter-marker gaps of >1Mb. Hence, a 100Mb genome would need about 1000 markers to be sure of producing a contiguous map. A typical fish genome would require 7000-10000 markers

If high-throughput methods for genotyping RH/Happy panels were available (oligo-microarrays, Illumina arrays) it might be feasible to have dense RH/Happy maps (>10000 markers)

Happy maps can be made for any organism (RH are likely limited to vertebrates)

BAC end sequences or contigs from low coverage whole-genome sequencing could be mapped on RH/Happy panels

Ron and Weller 2007

Minimal tiling path

Physical maps

BAC fingerprinting

Meyers et al. 2004 Nature Reviews Genetics 5: 578-89

BAC library Physical map RH panel/map

Cyprinus carpio (common carp) Katagiri et al. 2001 no no

Dicentrarchus labrax (European sea

bass)

BASSMAP BAC ends sequencing -

comparative mapping

Galibert unpublished

Gadus morhua (Atlantic cod) ? no no

Oncorhynchus mykiss (rainbow trout) Katagiri et al. 2001,

Palti et al 2004

? no (attempted?)

Scophthalmus maximus (turbot) no no no

Salmo salar (Atlantic salmon) Thorsen et al 2005 Ng et al 2006 no (attempted?)

Salmo trutta (brown trout) no no no

Solea senegalensis (Senegal sole) no no no

Sparus aurata (gilthead sea bream) BRIDGEMAP no Senger et al. 2006,

Sarropoulou et al. 2007

Acipenser spp. (sturgeons) ? no no

Crassostrea gigas (Pacific oyster) Cunningham et al

2006

no no

Mytilus edulis (blue mussel) no no no

Ostrea edulis (European oyster) no no no

Ruditapes philippinarum (Manila clam) no no no

Ron and Weller 2007

Single Nucleotide

Polymorphism (SNP) discovery

SNP genotyping

SNP discovery

Targeted re-sequencing (2 or more individuals) Known genesEPIC-PCRAnonymous genomic regions

Targeted sequencing di pooled DNAs

Random (shotgun) sequencing of genomic clones (or subgenomic libraries RRS reduced representation sequencing)

Random sequencing of ESTs of cDNA made with pooled RNAs + SNP discovery software

SNP discovery and genotyping (DNA microarrays?, ultra-high throughput sequencing?)

RRS

Endonuclease digestion and size selection

Genomic DNA from several individuals

Genomic sub-library

Shtogun sequencing

Sequence assembly

SNP discovery

SNP genotyping

Which method should we use?

Allele specific oligonucleotide hybridization

(ASO) (if array-based >100,000 SNPs can be

genotyped at once)

Primer extension (PE) (Mass Spectroscopy

allows cost-effective and limited

multiplexing genotyping)

Oligonucleotide ligation (OLA)

SNP frequency in the

genome

Candidate SNPs Validated SNPs Tools for high

throughput SNP

genotyping

Cyprinus carpio (common carp) ? ?

Dicentrarchus labrax (European sea bass) Type I 70-100 bp?

(Volckaert)

Type II 430 bp (Bargelloni)

591 (ESTs,

Volckaert)

69 (BAC ends)

70?

50

SequenomMassArray

(MGE)

Gadus morhua (Atlantic cod) ?

Oncorhynchus mykiss (rainbow trout) ? ? ?

Scophthalmus maximus (turbot) ?

Salmo salar (Atlantic salmon) Type I 614 bp Hayes et al.

2007

2507 (ESTs

mining)

86 SequenomMassArray

Salmo trutta (brown trout) ?

Solea senegalensis (Senegal sole) ?

Sparus aurata (gilthead sea bream) Type I Volckaert?

Type II 210 bp (Bargelloni)

618 (ESTs,)

55 (BAC ends)

10 ?

Acipenser spp. (sturgeons) ?

Crassostrea gigas (Pacific oyster) Type I 40 bp (Lapegue) MassSpec

(GoodAssay)

Mytilus edulis (blue mussel) ?

Ostrea edulis (European oyster) ?

Ruditapes philippinarum (Manila clam) ?

Ron and Weller 2007

Salmo salar (Atlantic salmon) 432,630Oncorhynchus mykiss (rainbow trout) 260,886Gadus morhua (Atlantic cod) 65,360Ictalurus punctatus (channel catfish) 44,767Cyprinus carpio (common carp) 19,364Oncorhynchus tshawytscha (Chinook salmon) 13,965Thunnus thynnus (Bluefin tuna) 10,163Penaeus monodon (giant tiger prawn) 7,484Mytilus galloprovincialis (blue mussel) 5,133Crassostrea gigas (Pacific oyster) 5,126Acipenser transmontanus (white sturgeon) 2,704Dicentrarchus labrax (European sea bass) 2,356Sparus aurata (gilthead sea bream) 2,282

Available ESTs in GenBank dbEST (31 August 2007)

Sparus aurata (gilthead sea bream)

Marine Genomics Europe >20,000 ESTs (from >10 libraries)Aquafirst >5,000 ESTs (from subtracted libraries, stress and disease)Wealth ?? (stress and disease?)Imaquanim ?? (disease, vaccination response)University of Padova > 5,000 (spleen)…..

cDNA arrays

spotted oligo DNA arrays

in situ synthesized oligo DNA arrays(different technologies, Affymetrix, Nimblegen, Combimatrix, Febit, Agilent….)

Agilent In Situ Oligo Synthesis

Glass Substrate

4 x 44 k probes on one slide

8x15k - 1x244k

Agilent Spike-Ins: Log(Signal) vs. Log(Relative concentration) Plot

Grid placement

cDNA libraries ESTs Array technology Reference

Cyprinus carpio (common carp) Many >22,000 cDNA CarpBase (Cossins

lab)Dicentrarchus labrax (European sea bass) Many >25,000 cDNA Canario Reinhardt

(MGE, Aquafirst)

Gadus morhua (Atlantic cod) Many >60,000

Oncorhynchus mykiss (rainbow trout) Many >250,000 cDNA, spotted oligo Prunet (INRA,

Aquafirst)

Scophthalmus maximus (turbot) 3 (disease) >12,000 Oligo array 3000

targets (15k probes)

Martinez U.Vigo

Salmo salar (Atlantic salmon) Many >400,000 cDNA GRASP

Salmo trutta (brown trout) ?

Solea senegalensis (Senegal sole) 10 (disease) 11,000 Oligo array (4200

targets)

Cerda

(Pleurogenes)Sparus aurata (gilthead sea bream) Many >30,000 cDNA

oligo array (19,000

targets)

Canario Reinhardt

(MGE, Aquafirst)

Bargelloni

Acipenser spp. (sturgeons) 3 13,000 - Giuffra (PTP Lodi)

Crassostrea gigas (Pacific oyster) many >20,000? cDNA Lapegue (MGE,

Aquafirst)

Mytilus edulis (blue mussel)

Ostrea edulis (European oyster)

Ruditapes philippinarum (Manila clam)

Coupled with 454 pyrosequencing (ultra-high throughput)

SuperSAGEarray (26nt probes on Febit oligo arrays)