scott williams ari wet lab manager former brood operations manager / genetics research clear springs...
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Scott WilliamsARI Wet Lab Manager
Former Brood Operations Manager / Genetics Research Clear Springs Foods
What causes disease in fish?
The Environmentcommercial trout culture
• Clear Springs Foods Brood Division– Two facilities
• Snake River / Research– Selection site– Brood propagation site– 5% of egg production
• Soda Springs– Egg production facility
Improving the hostselection goals
PrimarilyGrowth rate Indirectly for feed conversion rate
SecondarilyDisease resistance
Improving the Fish through Animal Breeding
Selecting the Parents of the Improved population
Using an Animal ModelGenetic merit based on:
Individual records Family records
Family and Within Family Selection for Size at 273 Days
• 100 to 110 full-sib families per generation• 10 families per generation from randomly bred line• Families selection:
– Families with poor early survival were discarded• Families with high mean weights at 273 days were
saved• Within the selected families:
– pit tag top 15 to 20% of individuals
Selection for GrowthSize at 273 Days
• 40 days Post Spawn, reduced to 600 individuals• 63 days PS, reduced to 100 individuals• 76 days PS, combined with another family in 100 gal
tank• 161 days PS, each family was given unique
clip/brand• 162 PS, moved to raceway• 273 PS, final measurement taken
Mean Weights at 328 Days PFodd year generation
0
200
400
600
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1600
1991 1993 1995 1997* 1999*
655701
908
1350
1524
497459
552
10231088
selected control
Mean Weights at 273 Dayseven year generations
Increasing size at 273 days post-fertilization
294
342
461
643
216236
217
437
669
450
0
100
200
300
400
500
600
700
800
1992* 1994* 1996 1998 2000
we
igh
t (g
)
CSF Control
Correlated traits questions:
• How does selection for rapid growth affect other traits?
• Conformation?• Fillet fat levels?• Disease resistance?
Genetics questionsrelated to disease:
• Is the ability to survive an IHNV infection a heritable trait, i.e., is it a trait that can be affected by selection?
• How does IHNV survival correlate to rapid growth?
Relationship of growth to IHNV Susceptibility
IHNV Mortality and size at 273 in BY 1999
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100%
450 500 550 600 650 700 750
Relationship of growth to IHNV Susceptibility
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IHNV Mortality and size in BY 2000
What is heritability?
A measure of the degree to which the variance in the distribution of a phenotype is due to genetic causes. Or simply, the degree to which genes control expression of a trait.
How heritable is the IHNV survival trait?
Estimate of the heritability is <0.83
This estimate confounds sources of genetic variation other than additive.
This appears to be a highly heritable trait though the actual value is lower than this estimate.
Heritabilities and Genetic Correlations Between Size and IHNV Susceptibility
size @ 63 d 0.399 0.716 -0.304challenge CPM 0.828 0.215size @ 273 d 0.405
size @ 63 d
challenge CPM
size @ 273 d
Fish with higher mean weights at the time of challenge, had greater CPM
Groups with higher ending weights had sibling with higher CPM rates
Tagged families from the 2000 year classfamilies tagged based 1° on IHNV resistance
0%
5%
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15%
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25%
30%
500 550 600 650 700 750 800 850
Per
cent
age
of a
ll fa
mili
es
Mean family weights
all families
selected families
Are there other traits correlated to disease resistance that could be
more easily measured?
Parental Humoral Response
humoral response - The immune response involving the transformation of B cells into plasma cells that produce and secrete antibodies to a specific antigen.
Traits Correlated to Disease Resistance: Parental Humoral Response
• Parents of the selected fish were PIT tagged• Eight months prior to spawn
– fish were IP exposed to attenuated form of the IHN virus.
• Six months later months later– Blood samples were collected from broodstock.– Samples and tag ID were correlated– Samples were used to evaluate each fish’s ability
to produce anti-IHNV neutralizing antibodies• Humoral response
Do the parental humoral responses correlate to the offspring challenge
results?
Weak effect of maternal titer on offspring survival rates.
Lower titer in the female associated with higher mortality rate in the offspring.
Improved Measurement of Disease Resistance
At 63 days post spawn• Two challenges
– Coldwater disease challenge• Two replicates of 50 fish each• Subcutaneous injection of Flavobacterium
psychrophilum– IHNV challenge
• One replicate of 50 fish• water borne exposure to IHNV in a closed, oxygenated
system for 1 hour– 10,000 plaque forming units/ml– Volume of water that is 10 times the total weight of the fish
Improved Measurement of Disease Resistance
• Mortality were collected and recorded daily• Necropsies were performed on a fraction of
the mortality to verify that the challenge organism was the cause of death
• Total cumulative percent mortality (CPM) and mean days to death (MDD) were calculated for each replicate
Distribution of Cold Water Disease Mortality 2001
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10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Pe
rce
nt
of
fam
ilie
s w
ith
in s
tra
in
Flavobacterium psychrophilum mortality range
Flavobacterium psychrophilum CPM histogram for all strains
CSF selected
control
CSI
Arlee
Average CWD Mortality Rate by Strain
Strain Flavobacterium psychrophilum CPM
CSF Selected 45.5%a
Control 38.6%a
CSI 62.57%b
Arlee 53.1%ab
Distribution of IHNV Mortality Rates in 2001
IHNV CPM histogram for all strains
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IHNV mortality range
Per
cen
t o
f fa
mili
es w
ith
in s
trai
n
CSF selected
control
CSI
Arlee
Average mortality rate by strain
StrainFlavobacterium
psychrophilum CPM IHNV CPM
CSF Selected 45.5%a 56.8%a
Control line 38.6%a 75.7%b
CSI 62.57%b 79.1%b
Arlee 53.1%ab 52.3%a
Distribution of CPM rates for Selected line 2002
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5%
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25%
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Pe
rce
nta
ge
of
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mili
es
Cummulative Percent Mortality
2002 Selected Groups' CPM during IHNV and Fp challenges
IHNV Fp
Relationship between mortality rates 2001
y = -0.0165x + 0.6036R2 = 0.0003
p=0.87
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IHN
V C
PM
Flavobacterium psychrophilum CPM
Relationship between Fp and IHNV mortality
Relationship between Mortality rates in 2001
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IHN
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ort
alit
y
Flavobacterium psychrophilum mortality
Relationship between Fp and IHNV mortality within the control and selected lines
selected
control
Relationship between mortality rates 2002
y = 0.334x + 0.536R² = 0.104
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Fp
CP
M
IHNV CPM
Fp and IHNV mortality
Mortality rates within Control and Selected lines in 2002
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Fp
CP
M
IHNV CPM
Relationship between Fp and IHNV CPM for control and selected lines
selected families
control families
Selection Program Modifications
• 1º Family selection on disease resistance– Entire families were culled based on the
mortality rates of replicates challenged• IHNV• Coldwater disease (CWD)
• 2º Within family size– Saving the largest individuals within the
families with the best survival rates
Family selection decision based on mortality index
• Index based on mortality rates of both of the pathogens
• Families with highest index scores were saved for grow-out phase of evaluation
• All other families terminated after growth evaluation at 273 days
Assessing disease resistance &within family selection for size
273 days of age• Weigh every member of all remaining families• Return on day 274
– Pit tag largest 15% within each of these families
Directions for growth and disease resistance selection
• Livability in the selected populations appeared to be decreasing relative to the unselected control line
• Common occurrence for lines selected primarily for improved growth, e.g., poultry
Directions for growth and disease resistance selection
• 1° selection based on a survival index– Index takes into account survival traits
• 2° selection will be based on growth rate• Results:
– Improved survival to IHNV– However, reduced rate of weight increases
Identification of DNA Markers Associated With IHNV Resistance in Rainbow Trout
Develop a rainbow trout linkage map Find molecular markers linked to loci
affecting resistance to infectious hematopoietic necrosis virus in rainbow trout
Develop a breeding plan to increase IHN resistance in rainbow trout using the genetic markers (Marker Assisted Selection)
Identification of DNA markers• Two strain of rainbows
– One anadromous and with elevated resistance to IHNV– The other a domestic strain with good growth and low
resistance to IHNV• Produce F1 cross• Back-crossing members of each family (F1) with rainbow
trout produced an BC1 generation• Challenge with IHNV• Collect mortality and survivors• Use both AFLP & microsatellite markers• Look for polymorphisms associated with survival or
mortality
Marker Assisted Selection for Trout Improvement
Growth Disease Resistance
selective breeding
large & resistant
small large susceptible resistant
largeresistant
Molecular tools
DNA extraction
QTL analysis
Marker Assisted Selection• Theoretically MAS has the potential to
greatly accelerate genetic improvement livestock, plants and aquatic animals.
• Initial data from livestock have demonstrated that MAS can be more productive than traditional breeding, but some cases can be equally or less effective than selective breeding.
• Limited data from aquatic species.