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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