Genetic linkage maps and QTLS assosiated with viral disease resist

ance in fish

Nobuaki Okamoto

Tokyo University of Marine Science

and Technology

Phenotype and Genotype

A. Symbols used in a pedigreeA. Symbols used in a pedigree

Father Mother

Daughter Son Sexunknown

Pregnancy Abortion

Parentalconsanguinity

( Completely dpcumented )

Daughter Sonaffected

Probabyaffected

( Incompletely documented )

Possiblyaffected

( not documented )

Femaleheterozygote

B. Genotype and phenotypeB. Genotype and phenotype

Two alleles, blue

bl and red r , at a gene locus:

homozygote red

homozygote heterozygote blue blue/red

blue/blue blue/red red/red

blue dominant over redred recessive to blue

blue recessive to redred dominant over blue

Phenotype

Genotype

Segregation of Parental Genotype

A. Possible mating types of genotypes for two alleles A. Possible mating types of genotypes for two alleles 　　　　 ( bl dominanto over r )( bl dominanto over r )

bl / bl bl / r bl / r r / r bl / r bl / r

bl / bl bl / bl bl / bl r / r r / r r / r

1. 2. 3.

4. 5. 6.

B. Expected distribution of genotypes in offspring of B. Expected distribution of genotypes in offspring of parents with two alleles, A and aparents with two alleles, A and a

Aa aa AA Aa Aa aa Aa Aa

1 : 1(50%) (50%) 0.5 0.5

1 : 2 : 1 (25%) (50%) (25%) 0.25 0.5 0.25

(100%) 1.0

Aa aa

Aa Aa AA aa

AA 　 aa

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

Linkage and Recombination

A. Recombination with crossing-overA. Recombination with crossing-over

1.Cytologic event

Parentalchromosomes

Meiosis WithoutCrossing-over Crossing-over

Gametes

1

2

3

4

Not recombinant Recombinant

2. Genetic result

ParentalGenotype(heterozygousAa and Bb )

Locus A

Locus B

Meiosis

Gametes

Not recombinant ( same as parental genotype )

Recombinant ( new )

B. Linkage of a gene locus with autosomal dominant mB. Linkage of a gene locus with autosomal dominant mutation ( B ) with a marker locus ( A )utation ( B ) with a marker locus ( A )

1. Without recombination

2. With recombinationMarker

Mutation

Recombinant

Distance Between Two loci and

Recombination Frequency

A.A. Recombination frequency as a consequence of the Recombination frequency as a consequence of the distance of two locidistance of two loci

Parents1 2

×

Offspring1 2 3 4

3%Recombinant

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 B. Determination of the relative distance and equence of three gene loci by measuring the frequency of three gene loci by measuring the frequency of recombination recombination

1. Gene loci A, B, C of unknown distance

2. Test cross of homozygous parental genotypes

AB × ab

AB Ab bA abAC Ac aC ac BC Bc cB bc

× ×AC acBC bc

31%Recombinant 8%

Recombinant 23%Recombinant

3. Relative distance

Segregation Analysis with RFLP Markers

A. Autosomal dominantA. Autosomal dominant

1. Without recombination 2. With recombination

1 2 3 4 5 6 7 8 1 2 3 4 5 6

RecombinantDNA fragments of 2.1 and 1.6 kb size

2.1 / 2.1 2.1 / 1.6 1.6 / 1.6

Probe

2.11.6

B. Autosomal recessiveB. Autosomal recessive

1 2 3 4 5 6 7 1 2 3 4 5 6 7

4.02.6

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 t

o 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 (T

ransgenic programs)• GMO(Genetically Modefied Organisms)

(Preparation of analyzed family

Positional candidate gene approach

○A ○B

○D ○E

(Making a genetic linkage map)

(QTL analysis)

(Marker-or gene-assisted selection ( MAS or GAS ) and marker-or gene-assisted introgression ( MAI or GAI )

○C (Genetic information of the traits,

Tools for genomic analysis)

(Positional cloning,

Causative genes)

○F

○1

○2

○3

Breeding program via marker–or gene-assisted selection ( MAS or GAS )

• 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.

Genetic linkage map for the tool of QTL analysis

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 genet

ic

　　 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 “ ” phaseⅠ 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 “ ” phaseⅡ 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 “ ” phaseⅢ 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 marker- assisted 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 th

e

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) conti

gs

・ 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 (Waldbieser et al., 2001) l • 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) • Spawning time (Sakamoto et al., 1999). • Disease resistance

Viral disease IPN (Ozaki et al., 2001) IHN (Khoo et al., 2000*)

Parasitic disease (Bartholomew et al., 2003*)

Japanese flounder: • Disease resistance

Viral disease LCD (Fuji et al., 2002*)

* : Presented in conferences.

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

• 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)

　 Infectious Hematopoietic Necrosis (IHN)

• 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

IHNV Challenge

) )

Lymphocystis disease (LCD)-QTL

LCD diseased fishLCD diseased fish

• 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.

Lymphocystis disease(LCD) in Japanese flounder

♀ Resistant

B strain

F1 hybrid (BA)

♀ Resistant

Backcross family(BAA)

Analytical family of Japanese FlounderAnalytical family of Japanese Flounder

♂ Susceptible

A strain

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

QTL analysis of LCD resistantQTL analysis of LCD resistant (Poli.9-8TUF on (Poli.9-8TUF on LG LG 15)15)

Fish affected with LCD Healthy fish (did not have LC cells)

◆ ◆◆ ◆◆ ◆◆ ◆◆ ◆◆ ◆ ◆◆ ◆◆ ◆◆ ◆◆ ◇ ◇◇ ◇◇ ◇◇ ◇◇ ◇◇ ◇◇ ◇ ◇◇ ◇◇ ◇◇ ◇

B A BA

116

124130

121

bp

LCD resistant (Poli.9-8TUF on LG15)Conclusion

Locus

Genotypes / Phenotypes

Lod

score ＊ 1 % ＊ 2

(n=139)

130 inherited 　 130 not inherited

LCD ＋ LCD － LCD ＋ LCD－ 　 15 : 54 62 : 8

14.8 45

＊ 2; the amount of the total trait variance which would be explained by a QTL at this locus, as a percent.

＊ 1; Lod score of 14.8, corresponding to 1014.8: 1 odds that the locus is linked. ( P value; 6.1e -20 ) 　　　　　　　　　

　　　　　　　　　　　　　　

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Looking into the future with hope in the sea