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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)