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Genomic resource development in onion

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Genomic resource development in onion John McCallum *, Samantha Baldwin *, Roopashree Revanna *, Meeghan Pither-Joyce *, Mark Fiers *, Ross Crowhurst *, Richard ... – PowerPoint PPT presentation

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Title: 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
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