Bay Scallop Genetic Resources and Applications
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Transcript of Bay Scallop Genetic Resources and Applications
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Overview and Application of Bay Scallop Genomic
ResourcesSteven Roberts
Marine Biological Laboratory
Woods Hole, MA USA
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Bay Scallop
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-fishery product-aquaculture product-scientific model
-muscle biology-photoreception
-essential environmental component
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Outline• Metamorphosis
– DEG– ESTs (overview) [example Iodothyronine Deiodinase]
• Muscle Growth– Candidate Gene [example Myostatin]
• Other uses for ESTs– Environmental response– Population genetics and restoration evaluations
• Available markers
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MetamorphosisTrocophore -> D-hingeBetween day 9-20 larvae first become “competent” then metamorphose into adult form.
High Mortality
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Why study bay scallop metamorphosis?
• Evolutionary Perspective
• Determine suitable natural habitat for successful recruitment
• Baseline information for identifying abnormalities
• Optimize aquaculture production
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Approach
Comparative transcriptomic analysis of developing bay scallops prior to (D-hinge), during (pediveliger), and following metamorphosis (spat)
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Differentially Expressed Genes (DEG)
Primer Pair BPrimer Pair A Primer Pair C
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Differentially Expressed Genes (DEG)
“D”
hing
e
pedi
velig
er
spat
“D”
hing
e
pedi
velig
er
spat
** **
ArbitraryPrimers:
MW
ACP 22 ACP 29
“D”
hing
e
pedi
velig
er
spat
“D”
hing
e
pedi
velig
er
spat
ACP 27 ACP 28
a
bc
a= Heat shock protein 70
b= serine protease precursor
c= pheromone receptor
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Expressed Sequence Tags (ESTs) agtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattc
ttcgctgacacagtcgacagtcnagtacgtaccgatcgcgcgccagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattc
tcgctgacacagtcgacagtcagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctaagtacgtaccgatagcatgatgcatgcatgctagcagcattacgagctagcgcattcgcaagctannnggttctcgttgattcgctgacacagtcgacagtcn
mRNA cDNA
Data Processing
mRNA cDNA
mRNA cDNA
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ESTs
• Individual clones sequenced– D-hinge 671– Met 61– Set 932– Adductor 367– Gonad 58
• 8% sequences <200 bp• Average length - 719 bp• 970 singletons• 217 contigs
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ESTs
• A. irradians – ESTs 7057 – CoreNucleotides 85
• Contigs 845• Singletons 2746• Unique Seqs 3591
C.gigas granulocyteZap Express cDNA library
- 2646 ESTs sequenced and annotated
- 343 contigs and 1319 singletons
-1662 Unique Seqs
- >40% not identified previously
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Hind limbs, rapid differentiation
Fore-limb emergence to complete tail reabsorption
Acquisition of hypothalamic sensitivity to thyroid
hormones
Premetamorphosis Prometamorphosis Climax Postmetamorphosis
T3/T4
TSH
TRH
Frog Metamorphosis
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Iodothyronine Deiodinase
Type 2 ID enzyme required for production of active
thyroid hormone• Tissue Distribution in Adult Bay Scallops
– Endostyle
Mucus secreting pharyngeal organ that facilitates filter feeding. Iodine concentrating ability and can synthesize thyroid hormone.
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T-iso
Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CCMP459
T-iso
T-iso
T-isoT-iso
T-iso
T-iso T-isoT-iso T-iso
CCMP459
T-iso
CCMP459
T-iso
CCMP459
T-iso
CCMP459
T-iso
CCMP459
T-iso
T-iso
Deiodinase u (ai Du)
Deiodinase v (ai Dv)
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Interrelationship with External Factors
Diet: Pavlova (e.g. CCMP549) has over 3 times more (%) phenylalanine than Isochrysis
Differential uptake by algae of selenium. Selenium required for synthesis of deiodinases.
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Does myostatin function in a similar manner in shellfish as it does in mammals?
= ==
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Myostatin expression in fish
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Scallop Myostatin
0
50
100
150
200
250
300
Mantle Gonad Heart DigestiveGland
Gill Muscle
Tissue
Hea
rt 1
Hea
rt 2
Gill
1
Gill
2
Dig
estiv
e G
land
1
Dig
estiv
e G
land
2
Gon
ad 1
Gon
ad 2
Man
tle 1
& 2
Mus
cle
1 &
2
Real-time RT-PCR – Dual labeled probes
Northern Blot
At least 6 times higher expression in adductor muscle tissue
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Myo-Blast Experiment
Figure 5. PCR products from RNA from adult scallops (N=2) treated with MyoZap and not treated (contols). Differentially expressed genes are indicated with lower case letters.
Cyclin T
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Myo-Blast Experiment
Control Experimental
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Other uses of ESTs
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Enhancement
Broodstock
Condition
Spawn
xx
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SeedWellfleet
ChathamDennis
32101
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EST-SSR M26Wellfleet
ChathamDennis
40
1030
Falmouth
Chilmark
20km
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Wellfleet
Dennis
32101
Chatham
g340
m26
n391
s336
Diver (aug) Diver (nov) Diver (nov) Spat Bag
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Regional
N391G340
M26
N391G340
M26
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Other Bay Scallop Markers
• EST-SSRs
• “Conventional” Microsats (Poster)
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EST-SNPs
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Acknowledgments• USDA-NRI 2003-35206-12834• Barnstable County, MA - CCCE• Linda McCauley & Christina Romano
• TECHNICAL [mbl]
• Hyun-Woo Kim & Don Mykles• MYOSTATIN [csu]
• Chris Dickson & Adam Bisonette• MYOSTATIN [undergraduate students]
• Gary Wikfors & Mark Dixon• METAMORPHOSIS [nmfs]
• Gabriele Gerlach• GENETICS [mbl]
• Steve Tettlebach & Maria Kretzman• GENETICS New York [liu]