Gen Hua YUE
Temasek Life Sciences Laboratory (TLL), Singapore
DBS, National University of Singapore
SBS, Nanyang Technological University, Singapore
Genomic approaches to the improvement of disease resistance in fish
Email: [email protected]
Outline
• Status of breeding fish for disease resistance
• Molecular approaches for the improvement of disease resistance
• Breeding Asian seabass (e.g. for disease resistance)
Aquaculture Industry
Prevention of Diseases
2. Vaccination
3. Breeding for disease resistance
• Effective, however • Labor-intensive • Only for specific diseases
• Inheritable & long-lasting • Difficult • Costly
1. Proper management• SOP • Skilled persons • Responsibility
Traditional Breeding for Disease Resistance
Prof.XimingGuo
MSX: Multinucleated Sphere Unknown
• A lot of diseases
• Very difficult in measuring disease resistance
• Only a few success cases, such as
• Influences of environmental factors • Low genetic gain/generation • Some traits are difficult to select • Reduction of genetic variation
Shortcomings of Traditional Breeding
Marker-assisted breeding can solve these problems.
Marker-Assisted Breeding (MAS)Marker assisted selection (MAS) refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening. Precondition: DNA markers can reliably predict phenotype
Advantages of MAS
Simpler method compared to phenotypic selection For traits with laborious screening; may save time and resources
Selection at earlier stage Important for traits such as meat quality, disease resistance, sexual maturity
Increased reliability No environmental effects Can discriminate between homozygotes and heterozygotes and select single fish
Marker development/Reference genome
Genome/DNA sequences
MAS/GS (genomic selection)
Marker characterization/genotyping
MAS in breeding
Linkage mapping
QTL mapping
QTL verification
GWAS
Validation
GS in breedingBarcoding
Diversity/structure
Relationships
Parentage
Short term 1-2 Y Middle term 3-8 Y
Requirements for QTL Mapping and GWAS
• Populations where DNA markers and trait segregate
• Many DNA markers covering a whole genome
• Cost-effective and high throughput genotyping
• Statistical analysis to detect the effects of location of DNA markers
QTL: Quantitative trait loci; GWAS: Genome wide association study
Populations with Variations
Disease outbreak or challenging fish with a pathogen
Survivals Dead fish
Types of DNA MarkersR
elat
ive
prod
uctiv
ilty
(log
scal
e)
0
1
2
3
4
Year1960 1973 1985 1998 2010
MutationsAllozymesRFLPsRAPD/AFLPsSSRsSNPs
Nobel prize in 1993
PCR
next generation sequencing
SNP
Microsatellites
Genotyping DNA Markers
SNP Genotyping using SNP-chip
SNP-chip/microarray
Genotyping by Sequencing
NextSeq 500
Next-Generation Sequencing Super computer
Big data analysisSequencing: Cheaper & Faster
2.7 Gb, 11 years
Statistical Analysis
Marker-Assisted Breeding
F2
P2
F1
P1 x
> 1000 individuals
Quick growthSlow growth
Find out the associations between DNA makers and traits: QTL mapping & GWAS
Selection is based on DNA markers
MARKER-ASSISTED SELECTION (MAS)
Status of Genomics in Aquaculture
• Genome seq: > 30 species • DNA markers: developed in most aquaculture species • Linkage map: constructed in > 45 fish species • QTL mapping: conducted in > 25 fish species • MAS: performed only in a few species
Marker-Assisted Selection-1
Successful Examples of MAS
IPN: Infectious Pancreatic Necrosis
Marker-Assisted Selection-2
Successful Examples of MAS
Prof. Nobuaki Okamoto
MAS of lymphocystis disease resistant flounders has a market penetration rate of 35% in Japan in 2012.
Asian Seabass (Lates calcarifer)
- History of culture: > 20 years
- Quick growth and good meat quality
- Sexual maturation at the age of 3- 4 years,
- Sex reversal (male to female) at the age of 3-5 years
- Over 1 million eggs/spawning
- Very compact genome ( ~ 700 Mb): 24 chromosome pairs
- We have worked on it since 1998 in Singapore
1500g400dph
0g0pdh
1g45dph
15g80dph
100g140dph
Hatchery Nursery phase Pre-grow-out Grow-out
NodaVirus
Bigbelly
Iridovirus
Tenacibaculum maritimum
Benedenia parasites
Streptococcus iniae
Immersion vaccine Injection vaccine
Major diseases of Asian seabass
Modified from http://www.thefishsite.com/articles/1086/diseases-of-farmed-barramundi-in-asia20
Genomic Resources for Asian Seabass• DNA markers > 10 million SNPs
Marine Biotechnology (2001), Molecular Ecology Notes (2002), Aquaculture (2006, 2009), Plos One (2014), Fish and Shellfish Immunology (2014), Fisheries Research (2015), Conservation Genetics (2015), Sci Rep (2015), Mol Ecol (2016), BMC Genomics (2017)
• cDNA, BAC Libraries and RNA-seq Animal Genetics (2006) , BMC Genomics (2008), Plos One (2010), DNA Research (2011, 2013) • Linkage and physical maps Genetics (2007), BMC Genomics (2011), Plos One (2010, 2012), Sci Rep (2015) • Candidate genes and MicroRNA Animal Genetics (2006, 2011, 2012), Plos One (2011, 2013), Animal (2012) • Whole genome sequencing Plos Genetics (2016, Laszlo’s group) • QTL for important traits BMC Genomics (2006), Animal Genetics (2008), BMC Genomics (2011, 2013),
Marine Biotechnology (2011, 2013, 2014, 2016), Sci Rep (2016), DNA Res (2016), Marine Biotechnology (2017) Data sharing among groups
Selection of Fish Resistant to Big Belly Disease
Size of fishes affected (up to 5 g, 25-50 dph)
Obvious clinic sign: big belly
Mortality: up to 99%
in three batches (F2-S16 and F2-S17) of F2 fishes in March 2012
Selection for Resistance to Diseases
Bigbelly
NNV
Why do We need Parentage Analysis?
15 x 15
offspring
1. Pedigree information is essential for selective breeding.
2. Unlike livestock, fish offspring cannot be easily differentiated by physical or electronic tagging.
3. Determining the realized reproductive success and the attributes of individual brooders are important in breeding.
Mechanism of Parentage Analysis
2 0 4 2 1 2
1000
2000
3000
22 42 1 2
1000
2000
2 0 4 2 1 2
1000
2000
500100015002000
500100015002000
19 6 20 0 2 0 0 2 2 4
excluded non-excluded partially-excluded
(“match”) (half-sib)
Candidate parents
Offspring
32720-023 32720-021
32721-023 32721-013 32721-036
How to Conduct Parentage Analysis?
Detection PCR products
Tissues/DNA
PCR
10 DNA markers
Scoring genotypes
Data analysisBluGEN
Contribution of Different Families to Offspring before and after big belly disease outbreak
%
After disease
Before disease
• Uneven contribution of different families
• Some families are more resistant to big belly disease than others
Major findings:
Brooders and Offspring Resistant to Big Belly Disease
F2-S16 F2-S17 F2-Laszlo
549494 547389 548923
541651 545165, 544872
21 190 30
Brooders ID
Offspring number
Advantages of our molecular breeding method: One generation ahead of other methods and maintaining diversity
Betanodavirus in Fish• causes VNN (Viral Nervous Necrosis), which
Occurs in > 40 marine fish species worldwide and some freshwater species
• > 90% of mortality in Asian seabass larvae
29
Test varieties
and release
Phenotype (lines have already been genotyped)
Train prediction
model
Make crossesand advance generations
Genotype
Advance lines informative for
model improvement
New Germplasm
Line Development
Cycle
Genomic Selection
Advance lines with highest
GEBV
Model Training Cycle
GWAS and GS for Resistance to NNV
MAS/GS reduces cycle time & cost by reducing frequency of phenotyping
Training Population for QTL mapping/ GWAS
Breeding Population
Challenge Fingerlings with NNV
Molecular Breeding: Resistance to big bellyResults of GWAS for Resistance aginst NNV
Fig3.SNPsassociatedwithresistanceagainstNodadovirusdetectedbyGWAS
GWASwith44KSNPsin1000fish(550survivaland500dead)from45families
Precision of GS for Resistance to NNV with Selected SNPs
Molecular Breeding: Resistance to big bellyGS for Resistance to Nodavirus
FieldtestsonresistancetoBetanodavirus(NV)areongoing
PredictionmodelsforGSSelectioncandidates
Genotypingwith96x4=384SNPs
FishresistanttoNNV
A elite Asian seabass lineF2 line-2
Resistant to diseases (Big Belly)
F + M
Breeding F3Resistant to diseases (Big Belly, Nodavirus, Iridovirus)
F4 …(Field testing)
Field testing + optimizing GS model
Increase of Growth of Seabass
Reaches 500-900 g in 9 months post hatchReaches > 3 kg in 2 years post hatch
Increase of Omega-3 in Our Asian Seabass
Asian Seabass: a High Omega-3 Fish
700
Three elite Asian seabass lines established F2 line-1 F2 line-3F2 line-2Quick growth
F + M
Resistant to diseases
F + M
High Omega-3
F + M
Breeding F3 Breeding F3 Breeding F3
Hybrid 1-2Hybrid 2-3
Hybrid 1-3
Hybrid 1-2-3
Our Goals
Ensuring food security in Singapore
The Next Big Fish
Acknowledgements• TLL management • Marine Aquaculture
Center, AVA • Prof. Laszlo Orban • Prof. Jimmy Kwang • Prof. Wong Sek Man • Prof. Hew Choy Leong • Mr. Huan Sein Lim • Dr. Junhui Jiang • Sequencing facility • All lab members
Funding