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 4 th International Workshop ICRISAT, India Feb 19, 2014
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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
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.