Anna Goostrey

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Anna Goostrey Tony Hawkins
Transcriptional profiling and SNP analysis of the
pacific oyster, Crassostrea gigas resistance and
susceptibility to summer mortality stress
A Collaborative Centre of the
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Combined genetic and functional genomic
approaches for stress and disease resistance
marker assisted selection in fish and shellfish
Aim To identify genes expressed in association
with disease and stress resistance Characterise
selected genes by expression profiling Analyse
allelic differences by examining SNPs
characteristic of resistant or susceptible
families
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Why oysters?

• Summer mortalities in the Pacific oyster are a
  recurring problem.
• These mortalities have a significant monetary
  impact on shellfish producers.
• Summer mortality is especially problematic in
  juveniles and is associated mainly with high
  temperatures and the oyster reproductive period.
• Additionally two oyster pathogens, Herpes-like
  virus and Vibrio spp.have been reported, but
  neither associated systematically with summer
  mortality.
• There is a strong and consistent genetic response
  of families to environmental stress.

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Complexities of summer mortality

• Summer mortality may well be the result of
  complex interactions between host, one or more
  pathogens and numerous environmental factors.
• Divergent selection criteria were applied during
  a selective breeding programme and a strong
  genetic basis for survival was observed
• F2 oyster progeny were separated into two groups
  resistant (R) and susceptible (S)

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Specific project outline

• Identify differentially expressed genes between R
  S families close to summer mortality events
• Produce a micro array with selected genes
• Use experimental RNA to validate micro array and
  identify genes up and down regulated in R S
  families
• Examine genes in summer mortality related
  processes using real time PCR and promoter
  characterisation
• Screen for SNPs specific to R S families in
  order to develop QTL markers for selective
  breeding

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Sampling for SSH
19 C
May
June
MORTALITY
R
S
Sampling at two time points. Subtract between R
S. Separate libraries for gill, mantle edge,
muscle, heamolymph, digestive gland and gonads
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Genes of interest to PML

• PML scientists established that faster growth
  and longer survival in individual shellfish
  result at least in large part from slower
  intensities with which proteins are renewed and
  replaced within the body as a whole
• Protein turnover
• essential for life
• continuous and intense
• energetically expensive
• consistently linked with
  
  genetic
  polymorphism

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Genes of interest to PML

• Genetic reductions in protein turnover
  facilitate
• higher growth efficiency
• slower incorporation of contaminants
• longer survival when energy is limited, or
  metabolism inhibited or uncoupled
• greater potential scope for activity
• increased metabolic stability
• lower energy costs of acclimation

Hawkins, AJS (1991) Protein turnover a
functional appraisal. Functional Ecology, 5
222-233. Hawkins, AJS. Day, AJ (1996) The
metabolic basis of genetic differences in growth
efficiency among marine animals. Journal of
Experimental Marine Biology and Ecology, 203
93-115. Hawkins, AJS (1997) Population responses
to contaminant exposure in marine animals
influences of genetic diversity measured as
allozyme polymorphism. In JP Seiler, JL Autrup
and H Autrup (Editors) Diversification in
toxicology - man and environment.  Archives of
Toxicology, Supplement 20, 429-442. Hawkins, AJS
Day, AJ (1999) Metabolic interrelations
underlying the physiological and evolutionary
advantages of genetic diversity. American
Zoologist, 39(2), 401-411.
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Genes of interest to PML
Using partial sequence data from EST database
Gigas Base and degenerate PCR we will be looking
at genes involved in metabolic pathways such as
cathepsin B, cathepsin D, aminopeptidase-N,
leucine aminopeptidase, alanine aminotransferase,
phosphoglucose isomerase and glutamine
deydrogenase, M2 pyruvate kinase, Riken,
cytochrome P450, malate dehydrogenase, ornithine
decarboxylase, transglutaminase
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ESTs chosen for micro array
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ESTs chosen for micro array
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Specific project outline

• Identify differentially expressed genes between R
  S families close to summer mortality events
• Produce a micro array with selected genes
• Use experimental RNA to validate micro array and
  identify genes up and down regulated in R S
  families
• Examine genes in summer mortality related
  processes using real time PCR and promoter
  characterisation
• Screen for SNPs specific to R S families in
  order to develop QTL markers for selective
  breeding

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Specific project outline

• Identify differentially expressed genes between R
  S families close to summer mortality events
• Produce a micro array with selected genes
• Use experimental RNA samples for micro array
  hybridisations and identify genes up and down
  regulated in R S families
• Examine genes in summer mortality related
  processes using real time PCR and promoter
  characterisation
• Screen for SNPs specific to R S families in
  order to develop QTL markers for selective
  breeding

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Samples for micro array hyb.

• Three experimental sets
• Sediment position 70cm vs 10cm
• Hypoxia
• Bacterial challenge Pathogenic non pathogenic
• Hybridisations divided between three labs
• IFREMER Hypoxia Montpellier Bacterial
  challenge PML Sediment

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Specific project outline

• Identify differentially expressed genes between R
  S families close to summer mortality events
• Produce a micro array with selected genes
• Use experimental RNA to validate micro array and
  identify genes up and down regulated in R S
  families
• Examine genes in summer mortality related
  processes using real time PCR and promoter
  characterisation.
• Screen for SNPs specific to R S families in
  order to develop QTL markers for selective
  breeding

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Specific project outline

• Identify differentially expressed genes between R
  S families close to summer mortality events
• Produce a micro array with selected genes
• Use experimental RNA to validate micro array and
  identify genes up and down regulated in R S
  families
• Examine genes in summer mortality related
  processes using real time PCR and promoter
  characterisation
• Screen for SNPs specific to R S families in
  order to develop QTL markers for selective
  breeding

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Detection of SNPs
Polymorphism between individuals Y2 AA Z1 GG Z2 GG
Ac AA
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Detection of SNPs
Individual heterozygote AG
Individual homozygote AA
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Marker assisted selection

• Screen for linkage/segragation of SNPs to R and
  S families in F1 and F2 progeny.
• Develop primer extension screening to examine
  farmed oyster populations.
• Selection of breeding programmes to enhance
  resistance to summer mortality.
• Aid to sustainable aquaculture

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Collaborating Oyster Partners
IFREMER La Tremblade Genetics and Hatchery team
IFREMER Brest
National University of Ireland Galway
A Collaborative Centre of the
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