Applications of Genotyping-by-Sequencing

for Wheat Breeding and Genetics

Jesse Poland

Kansas State University

Aug 22, 2013 1

Next Generation Genomics and

Integrated Breeding for Crop Improvement

4th International Workshop

ICRISAT, India

Feb 19, 2014

Aug 22, 2013 2

Early Generation Testing

Prelim Yield testing

(thousands)

Replicated Yield testing

(hundreds)

Advanced Yield testing

(tens)

Years

Varieties (one)

Crossing

Un-adapted

Exotic Elite

The Breeding

Funnel

Aug 22, 2013 3

Early Generation Testing

Prelim Yield testing

(thousands)

Replicated Yield testing

(hundreds)

Advanced Yield testing

(tens)

Years

Varieties (one)

Crossing

Elite Un-adapted

Exotic

The Breeding

Funnel

Genomic Selection

F2 enrichment

(MAS)

Back cross conversion

Parent selection

Molecular Markers and Objectives

Single Locus Typing

- Target known genes

- Few loci (<10)

Marker assisted selection

Backcross conversion

Cost per data point

Whole-genome Profile

- Assay whole genome

- Many loci (thousands)

Genomic Selection (AM)

Background selection

Diversity study

Germplasm typing

Cost per sample

Aug 22, 2013 4

Why use sequencing for genotyping rather than array based methods?

+ Amazing developments in sequencing output

+ Very good for wheat where polyploidy and duplications cause

problems with hybridization/PCR assays

+ Polymorphism discovery simultaneous with genotyping

+ No ascertainment bias

+ Low per sample cost

- Complex bioinformatics

- Requires paradigm shift in

molecular markers

Aug 22, 2013 5

Genotyping-by-sequencing (GBS)

Genotyping-by-sequencing (GBS)

“massively parallel sequencing” = sequencing on Illumina

platform

“multiplex” = using DNA barcode (unique 5-10bp)

- unique DNA sequence synthesized on the adapter

- pool 48-384 samples together

“reduced-representation” = use restriction enzyme to capture only

the portion of the genome flanking restriction sites

- methylation-sensitive restriction enzymes

- Target specific (rare, low-copy) sites in genome

- PstI (CTGCAG), MspI (CCGG)

Aug 22, 2013 6

“…massively parallel sequencing of multiplexed reduced-representation

genomic libraries.”

Aug 22, 2013 7

Poland, J. A. and T. W. Rife (2012) Genotyping-by-Sequencing for Plant Breeding and

Genetics. Plant Gen. 5(3): 92-102.

Application of GBS:

Aug 22, 2013 8

Genomic Selection

Genomic Selection Needed:

1) Training Population (genotypes + phenotypes)

2) Selection Candidates (genotypes)

Aug 22, 2013 9

Heffner, E.L., M.E. Sorrells, J.-L. Jannink. 2009. Genomic selection for crop improvement.

Crop Sci. 49:1-12. DOI: 10.2135/cropsci2008.08.0512

Inexpensive, high-density genotypes

Accurate phenotypes

Using GBS for GS

Is genotyping-by-sequencing a suitable marker platform

for genomic selection?

CIMMYT Semi-Arid Wheat Screening Nursery (SAWSN)

N = 254, advanced lines

Replicated field trials, Cd. Obregon, Mexico

Aug 22, 2013 10

Poland, J., J. Endelman, J. Dawson, J. Rutkoski, S. Wu, Y. Manes, S. Dreisigacker, J. Crossa, H.

Sanchez-Villeda, M. Sorrells, J.-L. Jannink. 2012. Genomic selection in wheat breeding using

genotyping-by-sequencing. Plant Gen. 5:103-113. DOI: 10.3835/plantgenome2012.06.0006

Using GBS for GS

CIMMYT Semi-Arid Wheat Screening Nursery (SAWSN)

GBS: PstI-MspI, 96-plex

HiSeq2000 = 180M – 210M reads / lane

41,371 SNPs 35K

DArT markers (n = 1,729)

Ridge-regression (rrBLUP)

7 sets for field trials – grouping factor

for cross-validation

No full-sib lines in same trial

11

Endelman, J.B. 2011. Ridge regression and other kernels for genomic selection with R package

rrBLUP. Plant Gen. 4:250-255. DOI: 10.3835/plantgenome2011.08.0024

Poland, J., J. Endelman, J. Dawson, J. Rutkoski, S. Wu, Y. Manes, S. Dreisigacker, J. Crossa, H. Sanchez-Villeda, M.

Sorrells, J.-L. Jannink. 2012. Genomic selection in wheat breeding using genotyping-by-sequencing. Plant Gen.

5:103-113. DOI: 10.3835/plantgenome2012.06.0006

Prediction of wheat quality

Aug 22, 2013 12

CIMMYT elite breeding lines (n=164) Cycle 45 International Bread Wheat Screening Nursery (C45IBWSN)

Replicated yield tests

2009 & 2010

6 environments

One replication for quality testing

milling

dough rheology

baking tests

Best Linear Unbiased Estimate (BLUE)

Genotyping-by-sequencing

15,330 SNPs (imputed with MVN-EM)(rrBLUP)

Cross-validation (x100)

Training sets of n=134

Validation sets of n=30

- thousand kernel weight

- mix time

- pup loaf volume

Sarah Battenfield, KSU

Aug 22, 2013 13

650

700

750

800

850

900

950

650 700 750 800 850 900 950

Ob

serv

ed

(cc)

Predicted (cc)

Loaf Volumes

r =

0.54

1

2

3

4

5

6

1 2 3 4 5 6

Ob

serv

ed

(m

in)

Predicted (min)

Mixograph Mix Times

r =

0.67

Sarah Battenfield, KSU

Training

Population

Cross

valid

both

years

Cross

valid

both

years

Cross

valid

both

years 2011 2010

Training Size (n) 1138 995 712 995 712

Testing

Population

Cross

valid

both

years

Cross

valid

both

years

Cross

valid

both

years 2010 2011

Testing Size (n) 569 712 995 712 995

Prediction Accuracies ( r )

Test Weight 0.725*** 0.723*** 0.715*** 0.312*** 0.192***

Grain Hardness 0.513*** 0.510*** 0.495*** 0.005 0.056

Grain Protein 0.630*** 0.629*** 0.620*** 0.400*** 0.335***

Flour Protein 0.604*** 0.602*** 0.589*** 0.394*** 0.284***

Flour SDS

Index 0.666*** 0.666*** 0.661*** 0.433*** 0.461***

Mixograph Mix

Time 0.718*** 0.715*** 0.707*** 0.535*** 0.499***

Alveograph W 0.697*** 0.695*** 0.683*** 0.512*** 0.475***

Alveograph P/L 0.476*** 0.474*** 0.466*** 0.323*** 0.278***

Loaf Volume 0.638*** 0.634*** 0.625*** 0.358*** 0.333***

Prediction of wheat quality

Regional Performance Nursery

Established 1931

1992 – Present

39 Locations

80,000 Plots

350,000 Obs.

792 SRPN + 428 NRPN Entries

44,924 SNPs

3,966 SNPs > 80%

Feb 17, 2014 14

Trevor Rife, KSU

Genomic Selection Accuracy: SRPN

Feb 17, 2014 15

Trevor Rife, KSU

RPN Locations: Evaluating Environments

Feb 17, 2014 16

Trevor Rife, KSU

Feed the Future Innovation Lab for

Applied Wheat Genomics

Dec 2, 2013 17 www.wheatgenetics.org/research/innovation-lab

Aug 22, 2013 18

Application of GBS:

Characterizing genetic diversity

Characterizing genetic diversity

Aug 22, 2013 19

Tanksley, S.D., S.R. McCouch. 1997. Seed banks and molecular maps: Unlocking genetic potential

from the wild. Science 277:1063-1066. DOI: 10.1126/science.277.5329.1063

Aegilops tauschii

(2n=2x=14, DD)

Triticum urartu

(2n=2x=14, AA)

~ Aegilops speltoides

(2n=2x=14, SS)

Triticum turgidum

(2n=4x=28, AABB) Triticum aestivum

(2n=6x=42, AABBDD)

Hybridization Events Forming Modern Wheat

Aug 22, 2013 20

Eric Olson, Michigan State University

Wheat Genetic Resource Center:

Aegilops tauschii collection

• 531 unique accessions

• Physiological classifications

• Genotyped with GBS

Aug 22, 2013 21

Aug 22, 2013 22

-0.05 0.00 0.05

-0.1

0-0

.05

0.0

00.0

50.1

00

.15

WGRC Ae. tauschii Collection

PCA1

PC

A2

tauschii

strangulata

typica

meyeri

56 %

5 %

Genetic groups

contrasting to

morphological

characteristics

Aug 22, 2013 23

-0.20 -0.15 -0.10 -0.05 0.00 0.05

-0.1

5-0

.10

-0.0

50

.00

0.0

50.1

00.1

5

WGRC Ae. tauschii Collection

PCA3

PCA4

Azerbaijan

Afghanistan

Iran

Tajikistan

Turkey

Pakistan

tauschii

strangulata

typica

2 %

2 %

Genetic separation

of geographically

separated groups

Genetic Diversity: Wheat vs Ae. tauschii

Aug 22, 2013 24

-0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04

-0.1

0-0

.05

0.0

00.0

5

WGRC Ae. tauschii Collection

PCA1

PC

A2

Ae.tauschii

wheat

63 %

10

%

-0.10 -0.05 0.00 0.05 0.10 0.15

-0.2

5-0

.15

-0.0

50

.00

0.0

5

WGRC Ae. tauschii Collection

PCA3

PC

A4

Ae.tauschii

wheat

2 %

2 %Limited diversity in

elite breeding pool

Aug 22, 2013 25

Application of GBS:

Genetic Mapping

Mapping Resistance in Synthetic Populations

Dec 2, 2013 26

LOD 5% TRTTF = 4.2

LOD 5% QTHJC = 3.7

0

5

10

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A 4B 4D5A 5B 5D 6A 6B 6D 7A 7B 7DChromosome

LO

D s

core

Stem rust resistance to race TRTTF and QTHJC in the SynOpDH population

Sandra Dunckel, KSU; Eric Olson, MSU; Matthew Rouse, USDA-ARS CDL

Sorrells, M. E., J. P. Gustafson, D. Somers, S. Chao, D. Benscher, G. Guedira-Brown, E. Huttner, A. Kilian, P. E. McGuire, K. Ross, J. Tanaka, P. Wenzl,

K. Williams and C. O. Qualset (2011). "Reconstruction of the Synthetic W7984 × Opata M85 wheat reference population." Genome 54(11): 875-882.

Dec 2, 2013 27

Traci Viinanen, University of Chicago; Lee DeHaan, The Land Institute

“F2” population

T7DL7J#1S

pAs1:green

GAA:red

Th. intermedium

GISH

Sr44

High-density genetic

maps for any species

High-density Genetic Map: Thinopyrum intermedium

Aug 22, 2013 28

Application of GBS:

Marker Assisted Selection

“Spiked GBS” A unified open platform for single marker genotyping and

whole-genome profiling

Utilize 1% of sequencing lane for targeted amplicon

sequencing

• Cost effective

• High-throughput

• Flexible: Single set of barcodes combined with

locus specific primers

Aug 22, 2013 29

QC & QUANTIFY

NORMALIZE DNA

DIGEST

LIGATE

ADAPTERS

WHOLE GENOME PROFILE (GBS)

($10 – 20 PER SAMPLE1) SINGLE LOCUS GENOTYPING (TARGET AMPLICONS)

(~ $0.03 PER GENOTYPE2)

PCR AMPLIFY TARGETS WITH M13 BARCODE PRIMERS

POOL

SAMPLES PCR AMPLIFY

POOL

QC AND QUANTIFY QC & QUANTIFY

“SPIKE” AMPLICON

LIBRARY AT 1%

NEXT-GEN SEQUENCING

GBS BIOINFORMATICS PIPELINE

~198M READS

RAW SEQUENCING DATA

~200M READS

~50,000 MARKERS ON 96 INDIVIDUALS

0.5X COVERAGE ~10 MARKERS ON 384 INDIVIDUALS

500X COVERAGE

TARGETED AMPLICON BIOINFORMATICS PIPELINE

~2M READS

1THE ESTIMATED COST PER SAMPLE IS BASED ON THE NUMBER OF SAMPLES THAT ARE MULTIPLEXED INTO A SINGLE SEQUENCING RUN AND THE COST OF THE

SEQUENCING. PER SAMPLE COST OF $10 CORRESPONDS TO GENOTYPING 190 INDIVIDUALS IN A MULTIPLEX SEQUENCING RUN. 2ESTIMATED COST PER DATA POINT FOR GENOTYPING 10 MARKERS ON 384 INDIVIDUALS.

Aug 22, 2013 30

“Spiked GBS”: SNP genotyping

• 96 winter wheat accessions

• GBS library

• Amplify 4 SNP loci and add at 1%

• “Converted” KASPar Markers

(removed selective bp, add tail for barcode)

Aug 22, 2013 31

0 2000 4000 6000 8000 10000 12000

02

000

4000

600

08

00

01

000

0

BS00023148

allele cnt A

allele

cnt

B

0 1 2 3

01

23

Lr34_exon12kasp

allele cnt A (log10)

allele

cnt B

(lo

g10)

0.0 0.5 1.0 1.5 2.0 2.5

0.0

0.5

1.0

1.5

Lr34_exon22kasp

allele cnt A (log10)

allele

cnt B

(lo

g10)

Aug 22, 2013 32

Application of GBS:

Variety Identification and Typing

Variety Confirmation and Identification

Aug 22, 2013 33

Breeder Seed Foundation Seed Production

Confirm Variety Identify Variety

Mixed up seed? A tail of two samples…

Feb 17, 2014 34

EXTENSION SAMPLE 1 EXTENSION SAMPLE 2

REFERENCE PERCENT IDENTITY PERCENT IDENTITY

Sample1 100.0% 74.0%

Sample2 74.0% 100.0%

Doans 74.7% 74.5%

Dodge 76.6% 72.6%

Duke 73.7% 74.8%

Dumas 74.1% 75.0%

Duster 76.0% 78.7%

E2041 79.3% 76.1%

Eagle 79.0% 76.8%

Endurance 74.4% 99.8%

Enhancer 76.9% 77.3%

Everest 75.2% 76.4%

Expedition 75.9% 77.7%

Fannin 75.5% 76.2%

Fuller 98.7% 74.7%

G1878 75.2% 74.6%

Gage 77.4% 76.5%

Gallagher 75.6% 81.0%

Garrison 78.3% 77.2%

2 sub-samples from each lot

extracted DNA

genotyped (along with a larger panel)

GBS

47,076 DNA markers

< 5% heterozygous markers

pure line varieties

74% identical markers

different varieties

Compared to reference panel and

varieties identified as Fuller (#1) and

Endurance (#2)

Aug 22, 2013 35

Ravi Singh

David Bonnett

Yann Manes

Susanne Dreisigacker

Jose Crossa

Hector Sanchez

Shuangye Wu★

Josh Sharon

Ryan Steeves

Jared Crain

Sandra Dunckel

Trevor Rife★

Traci Viinanen★

Narinder Singh

Daljit Singh

Lisa Borello

Bikram Gill

Bernd Friebe

Sunish Seghal

Jon Raupp

Allan Fritz

Sarah Battenfield

Chris Gaynor

Ed Buckler

Rob Elshire

Jeff Glaubitz

Jean-Luc Jannink

Mark Sorrells

Jeffrey Endelman★

Julie Dawson

Jessica Rutkoski★

Nils Stein

Martin Mascher ★

Burkhard Steuernagel

Thomas Nussbaumer

Robbie Waugh

Hui Liu