Aa aa Aa Aa AA aa AA aa C. Phenotypes and genotypes in the

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Transcript Aa aa Aa Aa AA aa AA aa C. Phenotypes and genotypes in the

Genetic linkage maps and QTLS
assosiated with viral disease
resistance in fish
Nobuaki Okamoto
Tokyo University of Marine Science
and Technology
Phenotype and Genotype
A. Symbols used in a pedigree
Father
Mother
Parental
consanguinity
Daughter
Daughter
Son
Son
affected
( Completely dpcumented )
Sex
unknown
Pregnancy
Probaby
affected
( Incompletely documented )
Abortion
Possibly
affected
( not documented )
Female
heterozygote
B. Genotype and phenotype
Two alleles, blue
bl and red
r
, at a gene locus:
Genotype
homozygote
blue
heterozygote
blue/red
homozygote
red
blue/blue
blue/red
red/red
Phenotype
blue dominant over red
red recessive to blue
blue recessive to red
red dominant over blue
Segregation of Parental Genotype
A. Possible mating types of genotypes for two alleles
( bl dominanto over r )
1.
2.
bl / bl
bl / r
3.
bl / r
r/r
5.
4.
bl / bl
bl / bl
bl / r
bl / r
r/r
r/r
6.
bl / bl
r/r
B. Expected distribution of genotypes in offspring of
parents with two alleles, A and a
Aa
aa
Aa
1
(50%)
0.5
aa
:
1
(50%)
0.5
Aa
AA
Aa
Aa
Aa
1
:
2
(25%)
(50%)
0.25
0.5
:
aa
1
(25%)
0.25
AA
aa
Aa
Aa
(100%)
1.0
C. Phenotypes and genotypes in the offspring of parents
with a dominant allele A and a recessive allele a
Aa
aa
Aa
aa
Aa
AA
Aa
Aa
Aa
aa
AA
aa
Aa
Aa
Linkage and Recombination
A. Recombination with crossing-over
1.Cytologic event
2. Genetic result
Parental
chromosomes
Parental
Genotype
(heterozygous
Aa and Bb )
Locus A
Locus B
Meiosis
Without
Crossing-over
Meiosis
Crossing-over
Gametes
Gametes
1
3
2
4
Not recombinant
Recombinant
Not recombinant
( same as
parental genotype )
Recombinant
( new )
B. Linkage of a gene locus with autosomal dominant
mutation ( B ) with a marker locus ( A )
1. Without recombination
2. With recombination
Marker
Mutation
Recombinant
Distance Between Two loci and
Recombination Frequency
A. Recombination frequency as a consequence of the
distance of two loci
1
Parents
2
×
1
2
Offspring
3%
Recombinant
3
4
97%
Not recombinant
3%
( 3.03 )
The recombination frequency of loci A and B ( 3% ) corresponds to their distance
B. Determination of the relative distance and equence of
three gene loci by measuring the frequency of
recombination
1. Gene loci A, B, C of unknown distance
2. Test cross of homozygous parental genotypes
AB
×
AB Ab
ab
AC
×
ac
BC
× bc
bA ab
AC Ac
31%
Recombinant
3. Relative distance
aC
8%
Recombinant
ac
BC
Bc
cB
bc
23%
Recombinant
Segregation Analysis with
RFLP Markers
A. Autosomal dominant
1. Without recombination
1
2
3
4
2. With recombination
5
6
7
8
1
2
3
4
5
6
2.1
1.6
DNA fragments of 2.1 and 1.6 kb size
Recombinant
Probe
2.1 / 2.1
2.1 / 1.6
1.6 / 1.6
B. Autosomal recessive
1
2
3
4
5
6
1
7
2
3
4
5
6
7
4.0
2.6
Recombinant
Problems to get informations of
causative genes
Economically
important traits such
as disease tolerance,
faster growing, better
taste
Control mechanisms
or gene functions are
unknown or have not
been indentified.
Molecular genetic approaches for breeding programs
in aquaculture
(1) Soft-pass
• Selective breeding using molecular markers
• Highly complementary to traditional breeding
• Being escaped from inbreeding depression-it is possible
to make a cross between different populations, tracing
the genetic information of important traits with DNA
markers.
• Non-GMO(Genetically Modified Organisms)
(2)Hard-pass
• Creating a hybrid organism using gene manipulations
(Transgenic programs)
• GMO(Genetically Modefied Organisms)
Breeding program via marker–or gene-assisted
selection ( MAS or GAS )
Positional candidate gene approach
1
○
B
○
A
○
(Preparation of
analyzed family
2 (Making a genetic linkage map)
○
F
○
C
○
(QTL analysis)
D
○
(Genetic information of the traits,
Tools for genomic analysis)
3
○
(Marker-or gene-assisted selection ( MAS
or GAS ) and marker-or gene-assisted
introgression ( MAI or GAI )
E
○
,
(Positional cloning
Causative genes)
Genetic linkage map for the tool of QTL
analysis
• Genetic linkage map is like an atlas which shows
the locations of genes or DNA fragments
(microsatellite markers).
• Microsatellite marker is a signpost to help locate
genes that expresses the desired traits
• Markers related to traits show the distance from
the genes responsible for the desired traits.
Marker - or gene -assisted selection
(MAS or GAS)
• Genotypic information obtained by DNA markers
is used for selective breeding program to produce
progeny enriched for desired traits (marker-or
gene-assisted selection,
MAS )
• If causative genes are iden tified, gene
- assisted
selection (GAS) will be carried out as the best
model.
A Preparation of analysing family
(1) Decision of the priority in economically important traits (EIT) that we want
to choose. For example. disease resistance, meat quality, better food
conversion efficacy.
(2) Confirmation of the methods to measure phenotypic charactors.
(3) Preparation of a cross family to analyse EIT. Phenotypic and genotypic
charactors of the selected parants should be strongly considered.
1) Looking for phenotipically positive and negative strains
2) Spreding phenotypic variance by gynogenesis techniques and developing
extreamly positive and negative fish or lines. At least two sets originated
from genetically different male are needed because genetic
polymorphism
is requested in an analysing family. For example, a cross family will
make
with a combination of a positive from A set and a negative from B set.
B Making a genetic linkage map
(1) Roughly constructed map
Mapped microsatellite markers (200-300 loci) and other DNA
polymorphic markers (AFLP etc).
(2) Detailly constructed map
Mapped microsatellite markers (3000~4000 loci), ESTs (expressed
sequence tags), and structural genes, being also able to use the genetic
information from other animals.
C QTL (quantitative traits loci) analysis
(1) analysed a cross family (F2 or Back cross).
(2) Measured the recombination frequency between phenotype of
pedigree materials and alleles of DNA markers.
(3) QTL is generally estimated by the results of both sides of the locus.
(4) Accuracy of the QTL position depends on the existence of DNA
markers that are closer to it.
D
Marker-assisted selection (MAS) and marker-assisted introgression (MAI)
1. MAS
(1) DNA markers tightly linked to phenotypes in the available pedegree materials can be
used for marker-assisted selection.
(2) Distinguished three distinct MAS phases, depending on the accuracy of the
information available about the economical important traits (EIT).
MAS “Ⅰ” phase
An EIT has been located with respect to relatively distant flanking markers.
Associations between specific marker and EIT alleles hold within specific families only
and need to be re-established for each family to allow for MAS.
MAS “Ⅱ” phase
An EIT has been fine-mapped with respect to closely linked markers which are in
linkage disequilibrium with EIT. Assocoations between specific markers and EIT alleles
hold across the populatin and not be re-established for each individual family, there by
greatly facilitating the implementation of MAS.
MAS “Ⅲ” phase
This phase is the optimum and is achieved when the causal genes and mutations have been
identified. This is called gene-assisted selection (GAS).
2. MAI
Favourable EIT alleles can be moved from specific family to other populations by markerassisted introgression (MAI). The accuracy of MAI depends on the phases of MAS; MAS
“Ⅲ” phase is the most effective.
E
Positional cloning
Although going from a map location obtained by linkage analysis to the
cloning of the actual gene and identification of the csusal mutation
remains a very time-consuming and costly task, the importance of
cloning the genes greatly facilitate marker-assisted selection MAS “Ⅲ”
phase) and also provides fundamental information about the biology
underlying production traits.
F Current tools for genomic analysis
・High density marker maps (microsatellites and SNPs (Single
nucleotide polymorphisms))
・Whole-Genome BAC (bacterial artificial chromosomes) contigs
・ESTs (expressed sequence tags)
・High density cDNA microarrays
・Complete sequence of the genome
・FISH (fluorescent in situ hydridization)
These directly or comparatively provide useful informations to
estimate and/or isolate candidate genes of EIT.
Aquaculture species that genome reseach has been
carried out
(1) Genetic linkage map available
• Tilapia
(Kocher et al., 1998)
• Rainbow trout (Salmonid fish) (Sakamoto et al., 2000)
• Channel catfish
l
(Waldbieser et al., 2001)
• Japanese flounder
(Coimbraet al., 2003)
(2) Under construction/inverstigation
• Red seabream, Yellowtail, Carp.Goldfish, loach,
Ayufish, Oyster, Shrimp and Seaweed (Porphyra)
Marker-QTL linkage analysis of
economically important traits in cultured fish
Rainbow trout:
• Temperature tolerance
(Jackson et al.,1998)
(Danzmann et al., 1999)
. 1999)
(Sakamoto et al.,
• Spawning time
• Disease resistance
Viral disease IPN
IHN
Parasitic disease
(Ozaki et al., 2001)
(Khoo et al., 2000*)
(Bartholomew et al., 2003*)
Japanese flounder:
• Disease resistance
Viral disease LCD
* : Presented in conferences.
(Fuji et al., 2002*)
IPN-QTL
LOD score = rlog 10 (θ) + (n-r)log 10(1-θ) + nlog10 (2)
n= number of progeny, r= number of recombinants, θ= r/n
IHN-QTL
Infectious Hematopoietic Necrosis (IHN)
• first reported in 1953 (1967 in rainbow trout)
• acute viral disease of Salmonids
• caused by rhabdovirus (IHNV)
• no prevention or treatment
• million dollars of loss each year
(US, Canada, Japan and other countries)
IHNV Challenge
• Age : 70 days
• Weight : 2.5±0.5g
• Induced by - 104 TCID50/ml IHNV
-12ºC
- immersion for 1 hour
• No. of fish/group : 100
• Duration : 30 days
)
)
Lymphocystis disease (LCD)
-QTL
LCD diseased fish
Lymphocystis disease(LCD) in Japanese
flounder
• A causative agent of LCD is lymphocystis disease virus
(LCDV; the Iridoviridae family).
• LCDV develops LC cells (hypertrophied cells) on skin,
fins and/or mouth.
• Almost every year, LCD outbreaks in cultured fish in
Japan and the farmers economically suffer from this
disease.
Analytical family of Japanese Flounder
B strain
A strain
♀ Resistant
♂ Susceptible
F1 hybrid (BA)
♀ Resistant
Backcross
family(BAA)
LCD resistance test in Backcross family
• Period; Feb - May, 2001
• Place; Kanagawa Prefectural Fisheries Research Institute
• Fish; Fish that have been reared with UV-treated water
until infectivity trial
139 progeny (1 year-old fish) from the backcross family
were exposed to LCDV-contaminated water
• Phenotypes;
LCD+ : Fish which developed LC cells
(hypertrophied cells) on skin, fins and/or
mouth
LCD- : Fish which did not develop LC cells on
skin,fins and/or mouth
B A BA
Fish affected with LCD
◆◆◆◆◆◆◆◆◆◆◆ ◆◆◆◆◆◆◆◆◆
Healthy fish (did not have LC cells)
◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇◇
bp
130
124
121
116
QTL analysis of LCD resistant
(Poli.9-8TUF on LG 15)
LCD resistant
(Poli.9-8TUF on LG15)Conclusion
Locus
Genotypes / Phenotypes
130 inherited
130 not inherited
LCD+ LCD - LCD + LCD-
62 : 8
(n=139) 15 : 54
*1; Lod score of 14.8, corresponding to 10
14.8
Lod
score *1
% *2
14.8
45
: 1 odds that
the locus is linked. ( P value; 6.1e -20 )
*2; the amount of the total trait variance which would be
explained by a QTL at this locus, as a percent.
・
Looking into the future with hope in the sea