Hazelnut Breeding in the Genomics Era

Hazelnut Breeding in the
Genomics Era
Shawn A. Mehlenbacher
Dept. of Horticulture
Oregon State University
University of California at Davis
June, 2011
Hazelnut Breeding in the Genomics Era
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Hazelnut, a model for the Betulaceae
Breeding program - the driving force
Eastern filbert blight resistance
DNA markers, marker-assisted selection, mapping
Microsatellite markers and uses
Map-based cloning of Gasaway resistance gene
Incompatibility in hazelnut
Sequencing the hazelnut genome (and transcriptome)
HRM for marker-assisted selection
Hybrid Hazelnut Research Consortium, SCRI grant
Taxonomy of Hazelnut (Corylus)
Rosids:
Order Fagales:
Angiosperms, Eudicots,
Core Eudicots, Rosids
from tolweb.org
Why sequence hazelnut?
European hazelnut (Corylus avellana) –
a model for the Betulaceae
•  relatively small stature
•  small genome (~380 Mb)
•  diploid (2n = 2x = 22)
•  relatively short life cycle, ~5 years to first flowering
•  OSU breeding program
•  genetic linkage map
•  BAC library
•  diverse collection of ~800 Corylus accessions
•  amenity to transformation with Agrobacterium
Statistics from sequenced plant genomes. Carica
papaya
Arabidopsis
thaliana
Populus
trichocarpa
Oryza sativa
(japonica)
Vitis
vinifera
372
125
485
389
487
No. chromosomes
9
5
19
12
19
G + C content (%)
35.3
35.0
33.3
43.0
36.2
Gene number
24,746
31,114
45,555
37,544
30,434
Avg. gene length (bp)
2,373
2,232
2,300
2,821
3,399
Avg. intron length (bp)
479
165
379
412
213
Transposons (%)
51.9
14
42
34.8
41.4
Size (Mbp)
Ming, R. et al. 2008. The draft genome of the transgenic tropical fruit tree papaya
(Carica papaya Linnaeus). Nature 452: 991-996 (24 April 2008).
Expect for hazelnut: 30,000 -35,000 genes, each ~2500 bp with 3 introns.
Numbers of genes that encode domains similar to plant R proteins
in Populus, Arabidopsis and Oryza. (Tuskan et al. 2006)
Predicted protein domains
Letter code
Populus
TIR-NBS
TN
10
21
—
TIR-NBS-LRR
TNL
64
83
—
TIR-NBS-LRR-TIR
TNLT
13
—
—
CC-NBS
CN
19
4
7
CC-NBS-LRR
CNL
119
51
159
BED/DUF1544*-NBS-LRR
BNL
24
—
—
NBS-LRR
NL
90
6
40
NBS
N
49
1
45
Others
—
—
41
284
398
207
535
Total NBS genes
Arabidopsis
Oryza
Plant genomes contain hundreds of resistance gene-like sequences.
European hazelnut (Corylus avellana)
At Corvallis Repository,
825 accessions of Corylus,
429 of C. avellana
~ 100 more at OSU
www.plantyfolia.com/photos106/corylus_ens.jpg
http://caliban.mpiz-koeln.mpg.de/~stueber/thome/band2/tafel_005.jpg
www.funghiitaliani.it/Alberi/nocciolo/Corylus%2520avellana1.jpg
The distribution of Corylus avellana includes many climatic zones, but
commercial production regions are limited.
(Mehlenbacher, 2003)
Hazelnut Production (MT)
1.
2.
3.
4.
5.
6.
Turkey
Italy
Azerbaijan (est.)
United States
Georgia (est.)
Spain
504,000
116,500
35,000
27,000
25,000
18,000
71.2%
16.4%
4.9%
3.8%
3.5%
2.5%
Hazelnuts in Turkey
are grown on the Black Sea Coast.
Tombul is an important cultivar.
It has long, clasping husks and
small nuts, for kernel market.
Hazelnuts in Oregon
are mechanically harvested. Nuts of
Barcelona fall free of the husk at maturity. The large nuts are suited
to the in-shell market.
Hazelnut Breeding Objectives
A. Blanched kernel market (for chocolate, baked goods)
1.
2.
3.
4.
5.
Bud mite resistance
Round nut shape
High percent kernel
Precocity
High yield
5.
6.
7.
8.
Easy pellicle removal
Few defects
Early maturity
Free-falling nuts
B. Resistance to eastern filbert blight (EFB)
1. Simply inherited resistance ( Gasaway & others)
2. Quantitative resistance (e.g. Tonda di Giffoni )
Hazelnut Breeding Flow Chart
1.
2.
3.
4.
5.
6.
7.
8.
9.
Choose parents, make crosses
Grow in greenhouse
10. Plant replicated trials
Seedlings in field
11. (Mehlenbacher & Azarenko)
12. Evaluate nuts
13. Evaluate nuts
Evaluate a few nuts
14. Evaluate nuts
Evaluate nuts
15. Evaluate nuts
Layer, Evaluate nuts
16. Evaluate nuts,
Nursery, Evaluate nuts
summarize data
17. Release new cultivar
Breeding cycle: 8 years from seed to seed.
Release of new cultivar: 16-17 yrs after cross.
Obtaining Hybrid Seed
Growing Hybrid Seedlings (4000 planted per year)
First Stage of Evaluation – Original Seedlings
with removal of discards
Propagation of Selections by
Tie-Off Layerage; suckers form every year
Harvested layers are
weak. They are held in
the nursery for one year,
and then planted in the
orchard.
Second Stage of Evaluation –
in Replicated Trials
Releases
Cultivars with Quantitative EFB Resistance:
Willamette
- 1990 cross made in 1973
Lewis
- 1997 cross made in 1981
Clark
- 1999 cross made in 1982
Sacajawea
- 2006 cross made in 1990
Cultivars with Very High Resistance:
Santiam
- 2005 cross made in 1989
Yamhill
- 2008 cross made in 1990
Jefferson
- 2009
cross made in 1993
Pollinizers with Very High Resistance:
VR4-31, VR11-27, VR20-11, VR23-18
Gamma, Delta, Epsilon, Zeta
Eta, Theta
- 1990
- 2002
- 2009
Cultivar susceptible to EFB: Tonda Pacifica - 2010
Yamhill (OSU 542.102)
was released for
the kernel market
in 2008.
Buyers are
interested.
Jefferson (OSU 703.007)
was released in 2009
as an EFB-resistant
replacement for
Barcelona . It is now
the most widely planted
cultivar in Oregon.
Late-shedding
pollinizers Eta and
Theta released at
same time.
Eastern Filbert Blight
Fungus Anisogramma anomala, 2-year life cycle. Cankers girdle and kill branches.
Disease Inocula-ons in the Greenhouse 3 scions per selection
Disease Inoculations in the Greenhouse
Cankers 13-16 months after inoculation.
Very High Resistance to EFB
in Corylus avellana
Genotype
Gasaway
Zimmerman
OSU 408.040
Ratoli
Georgian 759.010
OSU 495.072
COR 157
Culpla
Medium Long
Origin
Washington
Oregon
Minnesota "Weschcke Sdlg"
Spain
Republic of Georgia
Russia
Finland
Spain
Geneva, NY, USA
Very High Resistance to EFB
in Corylus avellana (cont d)
Genotype
Crvejne
Uebov
Moscow Selections (5)
OSU 1187.101
Seedlings (2)
Seedlings (3)
Seedlings (2)
Origin
Cacak, Serbia
Cacak, Serbia
Russia
Russia (Holmskij)
Russia
Crimea, Ukraine
Republic of Georgia
(~2% of accessions show very high
resistance)
Very High Resistance to EFB
in other Corylus species
C. americana Rush , now in BC2 generation
C. americana Winkler , now in BC1 generation
C. heterophylla Ogyoo , now in BC2
generation
Segregation ratios indicate single loci, with
dominant resistance.
RAPD Markers Linked to EFB Resistance
Robust RAPD markers are easy to
score, useful in many populations.
152-800
MAS because:
-  16 months between inoculation
and canker development, and
-  desire to select for resistance in
absence of pathogen.
Seedlings with one or both markers
are planted in the field. Seedlings
lacking both markers are
discarded.
268-580
Assignment of EFB Resistance Loci
to Linkage Groups
(based on co-segregation with SSR markers)
Gasaway
LG 6
Ratoli
LG 7
Georgian OSU 759.010
LG 2
OSU 408.040
LG 6?
Culpla
LG 6?
Russian OSU 495.072
NA
(The last three are the topic of Brooke Peterschmidt s M.S.
thesis research. Sagar Sathuvalli is developing new SSR
markers for LG6).
DNA Extraction using Juice Press
high throughput needed for MAS, 200 per day easy
Fusco Rubra
Ruby
Albania 55
OSU 495.049
Cutleaf
OSU 495.072
Gasaway
Zimmerman
Finland 187
OSU 681.078
OSU 408.040
Barcelonner Z.
Sweden 627
Aurea
Pendula
Aveline d'Angleterre
Des Anglais
Goc
Syrena
Redleaf Group
Red Fortrin
Rote Zellernuss
OSU 026.072
UPGMA dendrogram
based on microsatellite marker data
shows four main groups:
Central European Group
Central European
Black Sea
English
Spanish-Italian
Black Sea Group 1
Black Sea Group 2
English Group 1
English Group 2
Warsaw Red
Henneman #3
270 accessions incl. 72 synonyms
(Gökirmak et al., 2009)
Spanish-Italian Group
Parentage indicated for 31 accessions.
Zeta
0.1
Map-Based Cloning
Two targets: 1. EFB resistance from Gasaway
2. S-locus (incompatibility)
BAC library
Jefferson (best selection in mapping pop n)
constructed by Amplicon Express (Pullman, WA)
12 Χ genome coverage
mean insert size 110 kb
www.genome.iastate.edu/edu/doe/fig13.gif
If DNA markers that flank the resistance gene are known, chromosome walking can
proceed from both ends. The result is a contig that contains the gene of interest.
0.7
0.7
Map-based cloning of Gasaway
resistance
RAPD Markers on 6R:
5211200
5211200, CAC-C005
268580, 726665
268580, 726665
5.1
5.1
36 linked, 4 show no
recombination with R
C8-280
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
H04860
H04
R, W07860
365, X01825, AA12850, V20800
R, W07365, X01825, AA12850, V20800
173500
173
152800500
152
H19650800
, O161250, X021140, 122825, 2751130, 335600
H19650, O161250, X021140, 122825, 2751130,
335600, AR18830
3.6
3.6
1.4
1.4
Y19640, I15850
Y19640, I15850
A04510
A04510
Use for fine mapping:
~ 1488 seedlings from
mapping population.
High-resolution Genetic Map
EFB resistance locus and new
markers developed from BAC
end sequences, in mapping
population.
Resistance from Gasaway .
New markers were developed by
V.R. Sathuvalli in his PhD thesis
research
Physical Map of the EFB resistance region Gasaway gene is in Contig4. The 3 BACs were
sequenced. Of the 37 predicted genes, 5 had 100%
support from RNA-Seq data. Two predicted genes are in
classes known to have disease resistance properties.
The next target: incompatibility
One S-locus, 34 alleles
Co-dominance in Stigmas
Dominance or Co-dominance in Pollen
Fluorescence Microscopy
If the same allele is expressed by the stigma
and the pollen, the cross is incompatible.
Incompatibility Testing
Incompatibility Testing using
Fluorescence Microscopy
Compatible
Incompatible
Excellent germination
Poor germination
Long parallel tubes
Short tubes, bulbs
9-2100d
-1075
0
4
5
7
8
10
13
14
19
26
P15-300d
070-1400
073-1400
L20-550
W12-650
R19-1150d
Y14-750d 607-420d
AD02-650 414-610
G17-250
480-525d
34
764-1050
50
53
59
63
65
68
G05-510d
I06-750d
S3 204-950d
345-1050d
169-1250
211-680
Old (left) and new (right) maps for hazelnut LG 5S
showing the S-locus and linked markers.
Sequencing the Hazelnut Genome
•  Todd Mockler (OSU Botany & Plant Pathology) and Illumina
•  Genomic DNA of Jefferson
(heterozygous)
•  cDNA from leaves, young bark, kernels,
germinating seeds, styles.
•  Will allow comparisons with Betula, Castanea,
Quercus, others in the order Fagales.
Jefferson hazelnut sequence resources to date
Pilot Assembly Results
Velvet -> MIRA
SOAPdenovo
•  333,492 contigs
•  1,290,023 scaffolds
•  contig space ~340 Mbp
•  scaffold space = ~408 Mbp
•  contig N50 = 1,354 bp
•  scaffold N50 = 648 bp
•  max contig = 24,827 bp
•  max scaffold = 37,589
Work In Progress
•  add long mate-pair sequence data
(e.g. 2kb, 5kb, 10kb mate-pair data) to improve
long-range contiguity of the assembly
• add sequence data from Roche 454 runs (?)
•  reconcile scaffolds/contigs with map information
•  annotate the draft genome, incorporating RNA-seq data
•  mine SNPs, SSRs and other polymorphisms
•  release data to the public
•  sequence additional cultivars
Goal: marker-trait associations and candidate
genes to facilitate breeding efforts
BLAST interface http://corylus.cgrb.oregonstate.edu:8080/
How to use the sequence info?
•  improved understanding
(disease resistance, incompatibility)
•  knowledge of genetic diversity,
manage collection, choose parents
•  choose seedlings based on marker-trait ass ns
When? 4000 seedlings vs. 400 selections / yr
•  for seedlings, markers must be high-throughput
and robust
•  single nucleotide polymorphisms (SNPs)
abundant in plant genomes
•  MAS less useful for traits with high heritability;
most traits in hazelnut have high heritability
High Resolution Melting (HRM) Analysis
•  SNPs abundant in hazelnut genome (~1 in 50 bases)
•  SNPs result in differences in melting temperature
•  PCR amplicons 80-200 bp including primers.
•  PCR, 44-50 cycles of with LightScanner® High Sensitivity
Master Mix (Idaho Technology). LCGreen® Plus binds to
double-stranded DNA.
•  Denature at 94C for 0:30, cool to 28C to reanneal, and
begin melting.
•  HRM in a 96-well plate LightScanner (Idaho Technology)
collecting data from 55–97°C at a ramp rate of 0.10°C per
second.
www.genequantification.
de
A/T lower melting temp
en.wikipedia.
org/wiki/
High_Resolut
ion_Melt
G/C highest
melting temp
Mismatch –
lowest melting temp
Hybrid Hazelnut Research Consortium
Oregon State University
Rutgers University
University of Nebraska – Lincoln
National Arbor Day Foundation
Goal: Expand hazelnut production to eastern North
America.
Challenges: Eastern filbert blight, climatic adaptation
Plant materials: American x European hybrids
Expansion of Hazelnut Production, Feedstock, and Biofuel Potential
through Breeding for Disease Resistance and Climatic Adaptation
1. Map-based cloning of Gasaway gene (OSU)
2. Sequence EFB pathogen genome, develop markers (Rutgers)
3. Characterization of hybrids, nuts and EFB response (Rutgers, Nebraska)
4. Evaluate Corylus climatic adaptation, phenology (Rutgers, Nebraska)
5. Collect diverse C. americana and use in breeding (Arbor Day, OSU)
6. SSR marker assessment of diversity in C. americana & hybrids (OSU)
7. Oil content and kernel composition (Nebraska)
8. Economic analysis for eastern North America (Rutgers)
9. Outreach activities (Arbor Day, Nebraska)
SCRI proposal tasks
Behavior of EFB-resistant
selections in New Jersey
Gasaway and VR 20-11 – a few cankers on small twigs,
some stromata, cankers expand slowly.
Zimmerman – a few sunken cankers, no stromata
OSU 759.010 (Georgian) – infected but < T. di Giffoni
Ratoli (Spanish)
– no infection
OSU 495.072 (Russian) – no infection
OSU 541.147
– no infection (C. americana resistance)
OSU 526.041
– no infection (C. heterophylla resistance)
American hazelnut
Corylus americana
native to eastern North America
http://project.bio.iastate.edu/trees/campustrees/
images/Corylus/Corylus.jpeg
www.horticopia.com/hortpix/pix/U6HEH0.jpg
C. americana is expected to provide climatic adaptation
and EFB resistance.
Map after Drumke (1964)
UPGMA dendrogram of 162 accessions based on 21 SSR loci shows that
most hybrids were placed in one of three groups: Rush , ADF Group 1,
and ADF Group 2 (with Winkler ).
Sathuvalli, V.R. and S.A. Mehlenbacher. 2011. Characterization of
Americana hazelnut (Corylus americana) accessions and Corylus
americana x Corylus avellana hybrids using microsatellite markers.
Genetic Resources and Crop Evolution (submitted).
Ame531.016 MI Sorenson
Ame400.033 IA Peterson
Ame400.027 IA Peterson
Ame400.030 IA Peterson
Ame531.017 IA Peterson
Americana Group1
Ame405.060 MN Werner
Ame557.153 WI Prey
Ame400.040 WI Olejniczak
Ame531.038 WI Olejniczak
Mixed Group
ADF Hybrid Group2
Americana Group2
Ame401.030 WV Jones
Ame532.028 WV Jones
Hyb532.014 BAU
HybCOR638
AmePlymouth#3 IL White&Harris
Ame405.070 MO COR228
Ame405.079 MO Widrlechner
Americana Group3
Ame401.012 MD Thierny
Ame557.119 WI Boscobel
ADF14.123
Ame400.039 IA Grote
Ame531.027 IA Peterson
UPGMA dendrogram of Corylus americana groups 1 and 3. All
accessions are selected clones. The last two letters indicate the state
in which the seeds were originally collected. Fingerprints at 21 SSR
loci show great diversity among the C. americana accessions.
UPGMA dendrogram of
Corylus americana group 2
showing seven subgroups. All
accessions are selected
clones. The last two letters
indicate the state in which the
seeds were originally collected.
Fingerprints at 21 SSR loci
show great diversity among the
C. americana accessions.
Hazelnut Breeding in the Genomics Era
• 
• 
• 
• 
• 
• 
• 
• 
• 
Hazelnut, a model for the Betulaceae
Breeding program - the driving force
Eastern filbert blight resistance
DNA markers, marker-assisted selection, mapping
Map-based cloning of Gasaway resistance gene
Incompatibility in hazelnut
Sequencing the hazelnut genome (and transcriptome)
HRM for marker-assisted selection
Hybrid Hazelnut Research Consortium, SCRI grant
Thank you!