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: genhua@tll.org.sg

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