High-throughput SNP genotyping

for rice improvement

Dr. Michael J. Thomson

Senior Scientist, Molecular Genetics and Marker Applications,

Head, Genotyping Services Lab (GSL)

Plant Breeding, Genetics and Biotechnology Division

International Rice Research Institute, Philippines

5th International Conference on Next Generation Genomics and

Integrated Breeding for Crop Improvement

ICRISAT, India 18 February 2015

Genetic Resources

Plant Genomics

Plant Breeding

Marker development in rice

Diverse rice germplasm

“allele pool”

Beneficial alleles

Fine-mapping, candidate gene analysis, cloning

Association mapping Gene and QTL mapping

Flanking and gene-based markers

for molecular breeding

Genes controlling traits of interest

IRRI BREEDING PIPELINES

IRRIGATED RICE SOUTH ASIA

JAPONICA RICE

RAINFED RICE SOUTH-EAST ASIA

RAINFED RICE SOUTH ASIA

RAINFED / IRRIGATED RICE AFRICA

HYBRID RICE

IRRIGATED RICE SOUTH-EAST ASIA

IRRIGATED RICE SOUTH ASIA GREEN SUPER RICE

HEAT TOLERANCE

GRAIN QUALITY

SALINITY TOLERANCE

P/N USE EFFICIENCY

DROUGHT TOLERANCE

ANAEROBIC GERMINATION

YIELD POTENTIAL

COLD TOLERANCE

PHOTO-INSENSITIVITY

FLOOD TOLERANCE

DISEASE RESISTANCE

BIOFORTIFICATION (FE, ZN)

GREEN SUPER RICE

HEAT TOLERANCE

TRAIT DEVELOPMENT PIPELINES

GENE DISCOVERY

MARKER DEVELOPMENT

MOLECULAR BREEDING

QTL FINE-MAPPING

IRRI’s Plant Breeding and Trait Development

High-throughput SNP genotyping

Single nucleotide polymorphism (SNP) - a site in the DNA where individuals differ at a single base

SNPs

Haplotypes

Tag SNPs

Individual 1

Individual 2

Individual 3

Individual 4

SNP technology can accelerate genetic

mapping and allele mining

• Millions of SNP loci across the genome

• Most SNP markers are bi-allelic

• SNP data can be easily merged in a database

• Rapid high-throughput SNP genotyping systems are available

• SNP haplotypes can track specific alleles

Single nucleotide polymorphism

(SNP) marker – Infinium platform

Resources for SNP development in rice

OryzaSNP

resequencing

(McNally et al

2009 160k SNPs)

Sequencing

Genebank

(3,000 lines:

IRRI/CAAS/BGI)

Rice SNP

Consortium

(NGS on 125 lines:

16 million SNPs)

SNP

discovery

pools

44k SNP chip

(Zhao et al. 2011;

Cornell University)

High resolution

genome-wide

genotyping

700K SNP chip

(Cornell University

and IRRI)

6K SNP chips

(Infinium, several

developed)

2,015 KASPar assays (GCP/KBiosciences)

Custom 384-SNP sets (IRRI, Cornell, others)

High sample

throughput

genotyping

QTL mapping, genetic diversity

analysis, DNA fingerprinting

Trait-based diagnostic

SNP markers for breeding

Published

genes,

QTLs, and

SNP info

MAS

3,000 rice genomes

14x coverage

17 Tb of sequence reads

18.9M SNPs

IRRI CAAS

Raw data is available from GigaDb and SRA

N. Alexandrov, K. McNally, R. Mauleon, R. Hamilton, S. Tai, W. Wang, G. Zhang, Z. Li, and others

SNP-Seek database on the IRIC website:

http://oryzasnp.org/iric-portal/

International Rice Informatics Consortium

Querying SNP-Seek for sd1 LOC_Os01g66100

77 SNPs in 2235 lines

K. McNally

Initiative under the Global Rice

Science Partnership (GRiSP)

Product 2.1.3 “High-throughput SNP genotyping platform for breeding applications”

• Set up facilities for high-throughput SNP

genotyping

• Develop trait-based SNP markers for breeding

• Implement a SNP fingerprinting platform

http://gsl.irri.org

• Core facility at IRRI providing high-throughput SNP

genotyping services

• Serving IRRI and our regional partners

• >20,000 samples processed in 2014 providing 32

million SNP data points

Optimizing the DNA extraction

and genotyping workflow

DNA extraction:

Automated magnetic

bead system

Automated DNA

extraction

QC check:

Check DNA quality

and concentration

DNA quality

control

Leaf sampling:

PlantTrak sampling

into 96 well format

Tissue

sampling

with

PlantTrak

Data storage &

analysis:

SNP database/tools

SNP genotyping:

Custom Fluidigm and

Infinium sets of 24 –

4,600 SNPs

Fluidigm

24 or 96 SNPs

Infinium 6K chip

Supported by the “Transforming Rice Breeding” project

Barcode

scanner/leaf puncher Tissue sampling in the field using the PlantTrak Hx unit

96-sample plastic

magazines

Automated leaf sampling with PlantTrak

Brooks Automation

• Low cost minipreps and

high quality DNA

Automated DNA extractions on oKtopure

• Processes 8 x 96-well

plates in 1.5 hrs

Rapid plant growth for

molecular breeding

J. Chin and M. Thomson

• Set of RILs, NILs, designed

populations, improved varieties

with QTL/gene profiles

• MABC and QTL pyramiding

multiple trait packaging lines

with 3-, 4-, and 5-QTL

combinations in progress

Improved donor

development

Genotyping platforms for breeding applications

GoldenGate 384 SNPs

Fluidigm 24 and 96 SNPs

Infinium

6K SNP chip 4,600 SNPs

GBS 10k-40k SNPs

Fragment Analyzer

SSRs/indels Genetics and

Breeding applications:

Trait-based MAS,

Fine-mapping,

Rapid QC scans,

Background MABC,

Diversity analysis,

QTL mapping,

SNP fingerprinting,

High-resolution

mapping,

GEBV selection

Chr. 5 Chr. 8 Chr. 9 Chr. 11

0.43 0.48 0.57

0.92

1.40

1.97

SNP SNP SNP

SNP

SNP

SNP

0.04 0.15 0.22

SNP SNP SNP

25.68

26.35 26.45

26.72 26.90

27.34

27.79

28.30

SNP

SNP SNP

SNP SNP

SNP

SNP

SNP

25.07 SNP 5.30

5.92

6.22

6.40

6.75 6.91

7.33

8.15

SNP

SNP

SNP

SNP

SNP SNP

SNP

SNP

19.43

19.87

20.18

20.37 20.49

20.98

SNP

SNP

SNP

SNP SNP

SNP

18.87 SNP

Xa5

Xa13

Sub1 Xa21

Gene-based and flanking SNPs across Sub1 and Xa regions for

precise MABC selection

C. Vera Cruz, B. Collard, J. Chin

Validating trait-based SNPs for breeding

Validate trait-specific

SNPs predictive for

desired alleles

needed for breeding

programs

• Verify published

functional SNPs

• Test and optimize

functional SNPs on

Fluidigm system

• Develop “breeders’

chips” with custom

SNP marker

packages for target

traits

xa13 Deletion

base position

Adday sel

Makassane

NSIC Rc 222 222

Swarna-Sub1

Tong 88-7

NSIC Rc 238

MS 11

KHO

Dasanbyeo

Milyang 23

Tongil

TR 22183

N 22

Genetic variation within xa13 region

M. Dwiyanti, unpublished data

• Fluidigm Dynamic Arrays

for nano-liter reactions for

flexible SNP genotyping at a

low cost per sample

24 SNPs x 192 samples

(4,608 reactions)

• Validated functional and

gene-based SNPs for trait-

specific markers for breeding

Xa21 resistant allele

Xa21 susceptible allele

Trait-specific SNP markers for breeding

0.1

TN1TKM9

IRBL7 M CO IRBLk Ka CO

IRBLkp K60 CO

IRBL1 CL CO

IRBLkm Ts CO

IRBLkh K3 CO

IR 66946 3R 116

Brridhan47IRRI 147

IR 66946 3R 149IR4595 4 1 13IR 70023

Teqing

Minghui 63

MH63

IR06M143KaolackIR12T213

Aswina

AraiRaj

Supa

CAS209IRRI 132

IAC 165

Jumbot jetApo

IR 74371 70 1 1IRRI 148IR08L181

Mazan red

I geo tze

Zs97bCbb23

Khaohlanon

Khao mine lar

Sadu cho

Namwon1

Tetep

Taducan

Madabaru

Tsipala 421

Pokkali 108921

Pokkali

Rasht 454 1454

Hybrid line 2

Basmati 306

Rausrr 5

Laksmilota

Nanhi

N 22Vishunparag

Dz192

Kharsu 80a

Chengri

Kalshoni

Asepulujawa

Lien chan sha pu tsan

DV85

Dv 86

Aus 299

Chinsurahboro 2Ausboro

IRGC29086

DularAus 257

Aus 80BeriArc 11204

Hasawi irgc 16817 1FR13ARayada

Kalimekri 77 5

Tog175

Tupa 501

Tchampa

IRBL1 CLKay noi leuang

IR 67966 44 2 3

M202

Yrl 1

Koryeong13Unkwangbyeo

Tr41

Dasanbyeo 1

Ssalbyeo2

Toploea 70 76

Kamenoo

Tng67

Baghlani nangarhar

ToyonishikiMS11

Jinmibyeo

Taichung65Asominori

IRBLkp K60

IRBL5 M

IRBLz5 CA

IRBLkh K3

IRBLkm TS IRBLk KALTH

IRBLsh S

Tr22183 1

Tr22183

Rinaldo bersani

S4542a3 49b 2b12

N12

PI 298967 1

Cypress Gogo lempuk

Morobe

Chahora 144

Malagkit puti

Azucena

Hybrid line 1

Nova

Sorkheh zarrin shahr

Sadri ghermes Moosa tarom 57

MulaiTarem

TaremeMussataremValisehLaromeHassan tareme

Sadri32331

Anbarboo

Hashemi

Zardrome

Mussa taremeFirooz

Dom sufid

Sadri32339Sadri reza

Sherazi

SorkhrishekGhasib

Gharib

SadriGharibe

Rata 21 3

Begunbichi 348

Sal 104 ir 63 or sal 024Sambha mahsuri sub1Sambha mahsuri

IR 84196 12 32IR 84649IR11T153

IR12F566

Swarna sub1

Swarna

Br11

IR11F216Br11 sub1CR1009 Sub1

CR 1009

IR 55179IR4630

IR 4630 22 2 5 1 3

12ds gmet 22IR11A282

12ds gmet 3IR12A181BR29Br 29

IR11N202IR10F203Shiroudi

A 69 1FL478

IR29Hasawi

IR 66946 3R 178IR58443 6B 10 3IR58443IR 58443

Rasht 452 14932 IR12T217IR 85891

IR82252 145 2 3 3 2IR 2006

SundensisHanareumbyeo

IR24

UtrimerahIRBB13

IRBB5 IRBB59IR BB 60xIR 72920 1 44 4

IRBB54IRBB60IRBB63IRBB66

IR BB 66

IRBB52

IRBB55IRBB21IRBB62

IRBB7IRBB57

IRBB67IRBB65

IRBB64IRBB61

IRBB23

IRBB 23

IR71730 51 2

IRRI 163

IR 77674 3B 8 2 2 13 4 AJY2

IR 77674

IR 77674 3B 8 2 2 8 2 4

IR 77674 3B 8 2 2 12 5 AJY2IR 77674 3B 8 2 2 14 4 AJY1

IR 77674 3B 8 2 2 14 4 1IR07T118IR 77674 3B 8 2 2 8 2 AJY6

IR 77674 3B 8 2 2 14 2 AJY2IR 77674 3B 8 2 2 8 3 4IR 77674 3B 8 2 2 8 2 AJY10IR 77674 3B 8 2 2 12 5 1

IR 77674 3B 8 2 2 14 2 AJY4

IR 77674 3B 8 2 2 14 1 AJY5

IR09L120

IR 72046

Vandana 356

FEDEARROZ 50

IRRI119 RC68IRRI 165

IR12N135

BG1222

IR72890 81 3 2IR10N237

Nsic rc118

Matatag 1

IR10F550

IR10F548

IR11C115

IR11C114IR 45427 2B 2 2B 1 1

Shz2

SANHUANGZHAN NO 2

IR83614 1002 B BIR11T213IR 77674 3B 8 2 2 8 2 AJY4

Giza178

Nsicrc238

Nsicrc222

IR11F186

PSBRC18

NSIC RC 222

IRRI 154

IR12N253

IR10F365IR09F437

Psbrc18 sub1

IRRI 105

IR12F107

IR77664 B 25 1 2 1 3 12 3AJY1

Br 28

IR77542 551 1 1 1 1 2

IR06M139

IR11A306

IR11C170 IR11C169

IR 77674 3B 8 1 3 10 3 2

IR 6 PAKISTAN

Thadokkham 1

TDK Sub1IR09F185

IR57514 PMI 5 B 1 2

IR09F158

IRRI 119

IR09N534

IR09F171

IR09F166

IR09N496

IR03W134

IR11F211

IR10F109

IR12F164

IRBL9 W RL SPK2

IR 49830

Brri dhan 55IR10N271IR 29IR82489 594 3 2 2

IR12A207

IR10N225IR10A134

IR64 Heat 74

Brri dhan 53

IR07N112

IR64 Pup1

IR11T189IR64 Sub1 AG1IRRI149IRRI 149IR 64 SUB 1

IR64 AG1IR64 SALTOL

IR64 SPIKE

IR64 EMF

IR64 21IR64

IR64XGR2 R B3F8 239 19 4 20 11

IR64XGR2 R B3F8 239 19 9 13 32

IR64XGR2 R B3F8 239 28 6 3 7IR64XGR2 R B3F8 239 28 6 3 3

IR64XGR2 R B3F8 239 28 6 3 55IR64XGR2 R B3F8 239 10 16 11 36

Gr2 rxir64 b3f8 148 10 10 10 59

Gr2 rxir64 b3f8 148 10 10 10 19

Gr2 rxir64 b3f8 148 10 10 10 12

IR77298 5 6 18

IR10N238

Binadhan 712ds gmet 25IR10A231IR07A179IR09A136

IR 77298 14 1 2 10

IR11T222

IR09N212CiherangIR09F436Ciherang Sub1

IR06N155

IR06N119

IR11C138

IR83405 B B 96 2

IR78555 3 2 2 2

IR10N108

PSBRC 82

IR10F336IR11C173Rc82IR11C134IR05N412

IR04A115

IR11A303IR09N522IR09F154IR09F153IRRI 156IR09A228IR10F559

IR08N121IR10F571

Nsic 158

IR 77186 122 2 2 3IRRI 168

IR12T101

aus

indica

Temperate

japonica

Tropical

japonica

aromatic

Infinium 6K

chip for SNP

fingerprinting

and GEBVs

• 6K developed

at Cornell Univ.

(S. McCouch)

• 5,274 SNP loci

on 6K chip

• ~4,500 high-

quality SNPs

• 4,400 samples

run to date

• 6K data will be

used for

genome-wide

prediction

Over 6,000 samples run on the

6K SNP chip in 2014 providing

27 million marker data points

Genotyping by sequencing (GBS) for

GWAS on rice bacterial blight

• GBS can provide low-

cost high-resolution SNP

scans by multiplex

sequencing

• 96-plex GBS using ApeKI

per HiSeq lane (Cornell

University)

Elshire et al. 2011

• Testing GBS for

GWAS for resistance

to BLB disease

•285 diverse rice

accessions with 9

Xoo isolates

40K SNPs from GBS used for GWAS

• Known Xa genes (Xa4, Xa5, Xa21)

clearly detected in diverse panel

• Several potentially novel loci also

found

• GBS works well, but requires

significant data analysis

C. Dilla-Ermita et al

(unpublished)

Xa4 xa5

Integrating markers into breeding programs

MABC and

QTL pyramiding

IR64-

Sub1

IR64-

Pup1 IR64-

AG1

IR64-

heat IR64-

Saltol IR64-

DTY

MABC

lines

IR64-Pup1

-DTY-heat IR64-Sub1

-DTY-heat

IR64-Sub1

-AG-Saltol

QTL pyramid lines

MAS in the pedigree

breeding programs

F2

Varieties for different

target regions

F3

F4

F5

MAS

MAS

MAS

Yield trials

New released varieties

F2

F3

F4

F5

Genes/

QTLs

Causal variants

Genome-wide

prediction tools

Breeding

population

Genotyping

GEBV

Selections

Training

population

Genotyping/

phenotyping

Train GS

model

Testing and release

New released varieties

Thomson, 2014 (Plant Breeding Biotech. 2:195)

Breeding data management system

Breeding4Rice

Web-based

Information

Managemen

t

System

Facilitates

Study

Management

Use of Data

for Decision

Making

Interoperable

with other systems

such as Analytical

Services Breeding Information Management, IRRI. 2014

Genomic & Open-source Breeding Informatics Initiative (GOBII)

3 CG Centers:

Ithaca hub:

5 crop species: Maize, wheat, sorghum, chickpea, rice

• Susan McCouch

• Ed Buckler

• Jean-Luc Jannink

• Mark Sorrells

• Qi Sun

• Michael Olsen

• Rajeev Varshney

• Michael Thomson

• Lukas Mueller

GOBII APIs User Platforms

Indexing

Imputation

IBD calculation

Prediction

Selection

Mating design

High Density

Genomic Analysis

GOBII

QTL discovery

Segment

tracking

AP

I AP

I

IRRI-B4R

Pedigree records

Seed inventory

Experimental design

Data collection

User Interface

Data / Analysis

Selection

Mating design

MAS

Breeding

Scheme

KDDArT

Pedigree records

Seed inventory

Experimental design

Data collection

User Interface

Data / Analysis

Selection

Mating design

MAS

Breeding

Scheme

IBP-BMS

Pedigree records

Seed inventory

Experimental design

Data collection

User Interface

Data / Analysis

Selection

Mating design

MAS

Breeding

Scheme

DivSeek

Pedigree records

Seed inventory

Experimental design

Data collection

User Interface

Data / Analysis

Selection

Genetic, Trait,

Geographic info

Workflow optimization: • Ma. Ymber Reveche

• Socorro Carandang

• Annalhea Jarana

• Grace Cariño

IRRI Scientists:

J.H. Chin

R. Mauleon

E. Septiningsih

B. Collard

B. Zhou

C. Vera Cruz

K. McNally

E. Nissila

Acknowledgments

GSL team

Marker Validation: • Maria S. Dwiyanti

• C. Jade Dilla-Ermita

• Erwin Tandayu

• Crisostomo Dizon

GSL services: • Nadia Vieira Castañeda

• Geraldine Malitic-Layaoen

• Geisha Sanchez

• Venice Juanillas

• Krizzel Llantada

Genotyping Services Lab (GSL) team at IRRI

• Susan McCouch

• Mark Wright

Cornell

University

Funding: Japan Breeding project,

GRiSP program, Syngenta SKEP