Transcript Genomic resource development in onion - Main Page

The New Zealand Institute for Plant & Food Research Limited
Genomic resource
development in onion
John McCallum *, Samantha Baldwin *, Roopashree Revanna *, Meeghan
Pither-Joyce *, Mark Fiers *, Ross Crowhurst *, Richard MacKnight#
* Plant and Food Research, # University of Otago
Outline
• Developing resources to do
population genetic analyses in onion
Genetic stocks
Transcriptome
SNP and SSR markers
Informatics
Efficient typing
The New Zealand Institute for Plant & Food Research Limited
Onion (Allium cepa L.)
Most widely cultivated monocot outside of the grasses
Domesticated > 5000 years, unknown progenitor
Huge genome N=18 Gbp
~ 1 gene/BAC
2N=16 , out-crossing
Limited sequence
No synteny with grasses
The New Zealand Institute for Plant & Food Research Limited
Goals
• Understand crop genetic
structure and evolution to
enable conservation,
exploitation and seed QC/QA
• Understand genetics of bulb
quality and adaptation to
enable MAS
The New Zealand Institute for Plant & Food Research Limited
Family-Based Genetic Analysis in Allium
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Small population sizes to date
Mapping inefficient due to parental heterozygosity
Mapped major genes for fertility restoration, bulb composition
Good EST-based anchor marker
Comparative genomics stocks
• A. fistulosum /shallot alien addition lines
• A. cepa x A. roylei
• A. fistulosum
FF
FF+1A FF+2A FF+3A
FF+4A FF+5A
FF+6A
FF+7A
FF+8A
The New Zealand Institute for Plant & Food Research Limited
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http://alliumgenetics.org/
The New Zealand Institute for Plant & Food Research Limited
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Picture of onion field
Onion Populations
• Open-pollinated
landrace to elite
• Inbred limited mass
to S2
• Doubled Haploid not
significant in breeding
(yet)
• F1 hybrid based on 2
sources of CMS
• Shallot sexual and
asexual types
The New Zealand Institute for Plant & Food Research Limited
Within-population Genetic Diversity Of Onion
McCallum et al. J. Amer. Soc. Hort. Sci., 133(6), 810-818 (2008)
The New Zealand Institute for Plant & Food Research Limited
A. fistulosum
0.5
A. roylei
‘DH2150’
D H 2107
DDHH2110
DDHH2150
2122
2104
D H _ex_Y
IX _A
0.4
D H 2178
PCO of Jaccard
similarity matrix 289
peaks from 60 SSRs
0.3
MS U 611-1B
Shallot
B 1794B
Beltsville Bunching
0.2
MS U 2399B
B 1828B
P I_269415
N Y 53E 21B
C ayuga
0.1
Genetic Structure
in Onion
LC 2062
W 202A
MS U 826B
P I_177247
MS U 8155B
P I_174019
“Heterozygosity”
Texas _Grano_438
Iroquois
P I_179167
A C 43
P I_210994
0.0
P I_249901
P I_222228
A lfa_Tropical
Y _G_D anvers
P I_288274
-0.1
P I_239633
P I_243008
P I_274782
_C
reole
C M&
rop_E
RR
_P
arly_G
LK
Y
_S
_S p_W inegar
V iolet_de_Galmi
B Y G15-23
R _P inoy
Y IX _A
S enshu_K
i
ejo_H
LK ygro_F1
P I_168960
S outhport_W _G B P
P rimorska
P I_247067
P usa_W
hinese_Y
P I_235353
D owCning_Y
_G
Jumbo_
K iw i_Gold_
B aia_P erforme
P usa_Madhvi
S triginow
skiB eta_C ristal
Zittuaer_Glebe
A griFound_R
I_164970
B righam_YP_G
I_222764
PPI_181929
S
olara
ombay_R
N asik_R
S tuttgarter
PPPI_280554
A rka_NBiketan
I_207456
I_222698
I_251509
Italian_R
S outhport_R _G
K
alpatiya
oxa_B
arreiraP I_175571
C rioula R P
N
-53
Taherpuri
P I_271312
Whinese_P
_P ortugalurple
C
W _E benezer
W alla_W alla_S
‘Nasik Red’
P usa_R P oona_R
P I_271309
-0.2
-0.2
-0.1
-0.0
0.1
0.2
0.3
J. Amer. Soc. Hort. Sci.,
133(6), 810-818.
“domestication”
PCO2
The New Zealand Institute for Plant & Food Research Limited
‘Nasik Red x DH2150’
 Very wide cross
 Robust F1 & progeny
 Segregating for
multiple traits
frcfrc
120
180
240
300
360
420
480
Fructan mg/gDry Wt.
The New Zealand Institute for Plant & Food Research Limited
cDNA sequencing of ‘DH2150’ and ‘Nasik Red’
30000
25000
20000
ONGI V2.0
15000
count
• Normalised cDNA,
leaf+shoot
• ‘DH2150’
 Titanium run
 9.1 x 105 reads
 22 Mb assembly
• Nasik Red’
 GLX run
 5.8 x 105 reads
• BLAT to TIGR Onion
Gene Index reveals no
hits for 73% of contigs,
79% of singletons (E 10-5)
PFR Index 1.0
10000
5000
0
0
250
500
750
1000 1250 1500 1750 2000
length base pairs
The New Zealand Institute for Plant & Food Research Limited
SNP and CAPs Mining
• mapped ‘Nasik Red’
reads (72%) onto
‘DH2150’ reference with
Newbler
• 18,462 ‘high quality’
SNPs (one per 922 bp)
• designed primers
flanking putative
polymorphic HaeIII,
TaqI, PvuII, HinfI sites
using BioPython
pipeline.
The New Zealand Institute for Plant & Food Research Limited
Validation of SNPs conditioning HaeIII
polymorphisms between ‘DH2150’ and ‘Nasik Red’.
D- D+ N- N+
D- D+ N- N+
D- D+ N- N+
D- D+ N- N+
Current validation
rate ~ 50%
ACP127
ACP128 ACP129 ACP130
ACP133
ACP134
ACP135
ACP136
D- D+ N- N+
D- D+ N- N+
D- D+ N- N+
D- D+ N- N+
D=DH2150,
N=Nasik Red
- = no Enzyme
+ = plus enzyme
The New Zealand Institute for Plant & Food Research Limited
Genomic skim sequencing for gSSRs
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GLX genomic DNA library of ‘DH2150’ sequenced on 1/16 plate
29,786 reads, 6.6x106 bp of sequence
Newbler assembly, singletons screened
SSR detection + primer design using MISA Perl scripts
http://pgrc.ipk-gatersleben.de/misa/
 94/96 primer sets
amplified
26% amplify multiple
products
70% polymorphic in a
selection of 11 parental
lines including A.roylei
The New Zealand Institute for Plant & Food Research Limited
454-GLX Amplicon Re-sequencing
PCR with
Fusion A, B
barcodes
SNP Discovery Pilot
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√
23 Amplicons, 2 diverse pools
216 SNPs in 16 amplicons
Population Pilot
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»
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X
Ampure
cleanup
6 Amplicons,16 populations
MID barcodes
33 SNPs with large frequency diffs.
VA software obscure, misses
indels,no haplotypes, alignments
Haplotypes + freqs
VarScan
BLAT
SNPs + frequencies
R or
Python
QA/trim
Normalise &
pool
em-PCR + GLX run
fna +
qual
sff
Roche Variant
Analyzer
The New Zealand Institute for Plant & Food Research Limited
High-Throughput Phenotyping
Flow injection electro-spray MS (FIE-MS)
Chloroform/methanol/
water extract
N=96
plants
5 min direct infusion
Thermo-Finnigan MS
Low-res parent
ion masses +/‘Nasik Red x DH2150’ F2 Population
cdf format data
export
LC-Ms2
validation
R -analysis
FIEMSPro
PCA
The New Zealand Institute for Plant & Food Research Limited
PCA Biplot of m/z [M-H]-
sucrose
•Most influential PC1
ions confirmed by MSn
1-kestose
•Population also
segregates for bulb
fructan content
The New Zealand Institute for Plant & Food Research Limited
Using Zygem™ to Streamline MAS with a
Marker for the Onion R-locus
ACI023 genotype
Homozygous red
heterozygous
Homozygous brown
Stab bulb with
Pipette tip
Transfer tip to
microplate plate with
Zygem reagent
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b*
20
Incubate on
thermocycler
10
0
Take aliquot for
PCR
-10
-10
0
10
a*
20
30
The New Zealand Institute for Plant & Food Research Limited
Summary
NGS technologies - a godsend for obese and obscure
genomes!
Learning open-source tools - a worthwhile investment
We now have appropriate resources and systems to do
population genetic analyses of onion
The New Zealand Institute for Plant & Food Research Limited
Acknowledgements
Nigel Joyce & Martin Shaw (PFR) LC-MS and
biochemistry
Jo Stanton University of Otago High-Throughput
Sequencing Unit
FRST ‘Genes for Hybrid Seed’ C02X0803 and
‘Future Vegetables’ programmes
Enza Zaden NZ Ltd trialling,colour and Frc
genetics
Nunhems USA re-sequencing
Mike Havey (USDA-ARS) and Masayoshi Shigyo
(Yamagucho Univ.) –mapping collaboration
The New Zealand Institute for Plant & Food Research Limited