the new zealand institute for plant & food research limited genomic resource development in...

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


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


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


Family-Based Genetic Analysis in Allium

• 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


A. f

istu

losu

mA

. cep

a

1 2 3 4 5 6 7 8

http://alliumgenetics.org/


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


Within-population Genetic Diversity Of Onion

McCallum et al. J. Amer. Soc. Hort. Sci., 133(6), 810-818 (2008)


Genetic Structure in Onion PCO of Jaccard similarity matrix 289 peaks from 60 SSRs

Shallot

P I_207456

Jumbo_

P I_181929

P rimorska

P I_179167

Y _G_Danvers

P I_177247

W _P ortugal

P I_175571S tuttgarter

P I_174019

Y _S _S p_W inegar

P I_168960

S outhport_R_G

P I_164970

Roxa_B arreira

Y IX _AP LK

B Y G15-23

Italian_R

A C 43

Crioula

MS U826B

B righam_Y _G

MS U8155B

B eta_Cristal

MS U611-1B

A lfa_Tropical

MS U2399B

N-53

B 1828B

P usa_R

B 1794B

Nasik_R

W 202A

P usa_Madhvi

NY 53E 21B

R_P inoyB ejo_Hygro_F1

V iolet_de_Galmi

S olara

Cayuga

DH 2178

M& R_P LK

DH 2150

Texas_Grano_438

DH 2122

P I_288274

DH 2110

P I_274782

DH 2107

P I_271309

DH 2104

P I_251509

A.　roylei

B ombay_R

P I_247067B aia_P erforme

P I_239633

K alpatiya

P I_222764

P oona_R

P I_222228

Taherpuri

DH _ex_Y IX _A

A rka_Niketan

W alla_W alla_S

A griFound_RS triginowski

R_Creole

S enshu_K iCrop_E arly_G

LC 2062

Iroquois

Chinese_YK iw i_Gold_

P I_235353

Chinese_P urple

P I_210994

P I_280554

W _E benezerP I_271312

P usa_W

P I_269415

P I_243008

P I_249901

Downing_Y _G

P I_222698

S outhport_W _G

Zittuaer_Glebe

Beltsville Bunching

A.　fistulosum

0.4

0.2

0.0

0.3

-0.2

0.20.1-0.0

0.3

-0.1

-0.1

-0.2

0.1

0.5

PC

O1

PCO2

‘DH2150’

‘Nasik Red’

“domestication”

J. Amer. Soc. Hort. Sci., 133(6), 810-818.


‘Nasik Red x DH2150’ Very wide cross Robust F1 & progeny

Segregating for multiple traits

frcfrc


cDNA sequencing of ‘DH2150’ and ‘Nasik Red’

• Normalised cDNA, leaf+shoot• ‘DH2150’

Titanium run9.1 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)

150012501000750500

30000

250

20000

0

10000

20000

1750

25000

15000

5000

length base pairs

count

ONGI V2.0

PFR Index 1.0


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.


D=DH2150, N=Nasik Red- = no Enzyme+ = plus enzyme

D- D+ N- N+

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- D+ N- N+ D- D+ N- N+ D- D+ N- N+

Current validation rate ~ 50%

Validation of SNPs conditioning HaeIII polymorphisms between ‘DH2150’ and ‘Nasik Red’.


Genomic skim sequencing for gSSRs

• 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 amplified26% amplify multiple products70% polymorphic in a selection of 11 parental lines including A.roylei


454-GLX Amplicon Re-sequencingSNP Discovery Pilot

» 23 Amplicons, 2 diverse pools√ 216 SNPs in 16 amplicons

Population Pilot» 6 Amplicons,16 populations» MID barcodes√ 33 SNPs with large frequency diffs. X VA software obscure, misses

indels,no haplotypes, alignments

PCR with Fusion A, B

barcodes

Ampure cleanup

Normalise & pool

em-PCR + GLX run

sff

SNPs + frequenciesRoche Variant

Analyzer

VarScanfna + qualBLAT

R or Python QA/trim

Haplotypes + freqs


High-Throughput Phenotyping Flow injection electro-spray MS (FIE-MS)

Chloroform/methanol/water

extract

5 min direct infusionThermo-Finnigan MS

Low-res parent ion masses +/-

cdf format data export

‘Nasik Red x DH2150’ F2 Population

R -analysisFIEMSPro

PCA

LC-Ms2 validation

N=96plants


PCA Biplot of m/z [M-H]-

•Most influential PC1

ions confirmed by MSn

•Population also

segregates for bulb

fructan content

1-kestose

sucrose

The New Zealand Institute for Plant & Food Research Limiteda*

-10 0 10 20 30

b*

-10

0

10

20

30

Using Zygem™ to Streamline MAS with a Marker for the Onion R-locus

RR brown

Rr red

rr dark

red

Stab bulb withPipette tip

Transfer tip to microplate plate

with Zygem reagent

Incubate on thermocycler

Take aliquot for PCR

ACI023 genotype

heterozygous

Homozygous red

Homozygous brown


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


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 genomic resource development in...

Documents

new zealand institute

plant food research

limited sequence

fistulosum ff ff

mas slide

university of otago

elite inbred limited

mapped nasik red reads