Genomic resource development in onion

1
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

2
Outline

• Developing resources to do population genetic
  analyses in onion
• Genetic stocks
• Transcriptome
• SNP and SSR markers
• Informatics
• Efficient typing

3
Onion (Allium cepa L.)

• Most widely cultivated monocot outside of the
  grasses
• Domesticated gt 5000 years, unknown progenitor
• Huge genome N18 Gbp 1 gene/BAC
• 2N16 , out-crossing
• Limited sequence
• No synteny with grasses

4
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

5
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

6
http//alliumgenetics.org/
7
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

8
Within-population Genetic Diversity Of Onion
McCallum et al. J. Amer. Soc. Hort. Sci., 133(6),
810-818 (2008)  
9
Genetic Structure in Onion PCO of Jaccard
similarity matrix 289 peaks from 60 SSRs
DH2150
Heterozygosity
Nasik Red
J. Amer. Soc. Hort. Sci., 133(6), 810-818.  
domestication
10
Nasik Red x DH2150

• Very wide cross
• Robust F1 progeny
• Segregating for multiple traits

frcfrc
11
cDNA sequencing of DH2150 and Nasik Red

• Normalised cDNA, leafshoot
• 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)

12
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.

13
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
DDH2150, NNasik Red - no Enzyme plus
enzyme
ACP133
ACP134
ACP135
ACP136
D- D N- N
D- D N- N
D- D N- N
D- D N- N
14
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 amplified
• 26 amplify multiple products
• 70 polymorphic in a selection of 11 parental
  lines including A.roylei

15
454-GLX Amplicon Re-sequencing
PCR with Fusion A, B barcodes

• SNP 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.
• VA software obscure, misses indels,no haplotypes,
  alignments

Ampure cleanup
Normalise pool
em-PCR GLX run
sff
SNPs frequencies
16
High-Throughput Phenotyping Flow injection
electro-spray MS (FIE-MS)
Chloroform/methanol/water extract
N96 plants
5 min direct infusion Thermo-Finnigan MS
Low-res parent ion masses /-
Nasik Red x DH2150 F2 Population
cdf format data export
R -analysis FIEMSPro PCA
LC-Ms2 validation
17
PCA Biplot of m/z M-H-

• Most influential PC1 ions confirmed by MSn
• Population also segregates for bulb fructan
  content

sucrose
1-kestose
18
Using Zygem to Streamline MAS with a Marker for
the Onion R-locus
Stab bulb with Pipette tip
Transfer tip to microplate plate with Zygem
reagent
RR brown
Rr red
rr dark red
Incubate on thermocycler
Take aliquot for PCR
19
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

20
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