the new zealand institute for plant & food research limited potato genome sequencing consortium,...

The New Zealand Institute for Plant & Food Research Limited

Potato Genome Sequencing Consortium, notes from the edge

Dr Susan Thomson, Dr Mark Fiers, Dr Jeanne Jacobs


Potato Genome Sequencing – why?

Solanaceae - important family (tomato, eggplant, petunia, tobacco, and capsicum)

Potato is now the 3rd largest global food crop


Potato Genome Sequencing – the beginning

The Potato Genome Sequencing Consortium is an initiative of Wageningen University & Research Center

PGSC brings together a global community to complete the project.

Individual partners were assigned different chromosomes.


PGSC – member countries


PGSC – the beginning

1995 – Genetic map of potato, diploid mapping populationSH (SH83-92-488) x RH (RH89-039-16)


Genetic map 1995

2001 – Ultra High Density genetic map generated,~10,000 AFLP markers (genome ~840Mb, 12 markers/Mb)



Ultra high-densitygenetic map

2001

Genetic map 1995

2002 – BAC library, using RH89-039-16.85,000 BACs average insert of 120Kb.73,000 fingerprinted by AFLP




2001

BAC library2002

Genetic map 1995

2006 – AFLP analysis of BACs used to build up contigs of overlapping BACs. Selective AFLPs used to anchor certain BACs (and contigs) to physical map.




2001

BAC library2002

Genetic map 1995

Physical map2006

2005/6 – Initiate genome sequencing. BAC by BAC Sanger sequence. Start with anchored seed BACs.6x coverage, 800- 1000 BACs/chromosome.




2001

BAC library2002

Genetic map 1995

Physical map2006

Dec 2009 – end date for full annotated potato genome sequence

Sequencing start

2005/6



Sequencing start

2005/6


Annotation and sequence Dec 2009

Early 2008 – BAC sequencing status: chromosome 7 not started, others very few BACs done.


PGSC – the worries

Sanger BAC by BAC slow Despite UHD map of 10,000 markers,still large gaps in physical map reducing number of seed BACs

Made more problematic by ‘stops’ caused by repeat elementsand lack of overlapping BACs


PGSC – the solutions

Bigger and better machines!

Next Generation Sequencing (NGS) technologies making Whole Genome Shotgun (WGS) sequencing more financiallyfeasible (data/$).

RH is highly heterozygous, leading to assembly issues.

Continue RH sequencing using mainly NGS methods


PGSC – the solutions

Introducing a new line, DM: DM 1-3 516 R44

Doubled Monohaploid, homozygous, line.(Ref: Lightbourn GJ, Jelesko JG, Veillieux RE. 2007. Genome 50 (5):492–501.)

DM flowers well.

Can be used as female parent in crosses with most diploid potato germplasm.


PGSC – mapping

No genetic knowledge for DM 1-3 516 R44

Diploid mapping population:

DM x DI (China Runtush)

F1 x DI

Mapping population

2 x 96 well plates with DNA of mapping population, along with parents.Generated by International Potato Center (CIP), Peru.


PGSC – mapping

Preliminary Scaffold assembly of DM derived from Illumina data:(generated by Beijing Genome Institute, BGI)

No. of sequences 57681 max scaffold length 5398072 min scaffold length 100 total assembly length 702581734 average scaffold length 12180 median scaffold length 179 n50* 429749

* n50 = largest first, align along length of genome. n50 is size of scaffold at 50% genome.

As at 20 August 2009


PGSC – mapping

550 newly generated SSR markers*; SSRs Institute Country 100 Plant and Food Research New Zealand 100 Universidad Nacional Agraria La Molina Peru 100 International Potato Centre Peru 100 Scottish Crop Research Institute Scotland 50 Instituto Nacional de Tecnologia Agropecuaria Argentina

50

The Irish Agriculture and Food Development Authority, Teagasc Eire

50 Institute of Bioengineering Russian Federation

*SSRs generated by BGI, China

Preliminary results 14/44 were monomorphic, 15/44 tested show polymorphism in DI, 15/44 show polymorphism between DM/DI


PGSC – mapping

- 148 Sequence Tagged Markers (STM). Known to map to regions spanning all 12 chromosomes.

- ~60 Ste markers, currently being mapped in an SHxRH population. Generated by large scale in-silico design of SSRs from ESTs in public database. (Ref: Tang J, Baldwin SJ, Jacobs JM, Linden CG, Voorrips RE, Leunissen JA, van Eck H, Vosman B. BMC Bioinformatics. 2008 Sep 15;9:374)


PGSC – mapping

SNP data – EST data aligned to DM scaffold. (Robin Buell, courtesy of SolCAP USDA project http://solcap.msu.edu/)

Design ~ 2000 markers for use with BeadXpress (Illumina) (Glenn Bryan, Scottish Crop Research Institute)

Aiming for > 1000 mapped.

DArT data*– Two discovery arrays with over 30,000 probes to begin. Discovered 3000 candidate markers.

It is hoped that 1000 to 1500 unique DM markers will segregate in the mapping population.

Sequencing of 7000 DArT markers will also be carried out.

* Diversity Arrays http://www.diversityarrays.com/

Mapping data will be combined with results from:


PGSC – assembly

Plans for an in silico* pipeline to improve scaffold bringing together data from:

- SOL Genomics Network

- Tomato genome

- Markers; SSR, SNP and DArT

- RH UHD/physical map information

* Dan Bolser, University of Dundee, Scotland


PGSC – the present & future

Line In progress Sanger sequencingIllumina

runsRoche/454

runs

RH WGS + Long Jump libraries

10 X coverage

WGS 60 X coverage

BAC library150,000 BAC end sequences + 2,000 BAC clones

Random sheared BAC library (~100kb) 120,000 BAC end sequences

DM WGS + Long jump libraries

10 X coverage

WGS + 500bp to 10kb libraries

65 X coverage

Fosmid library (~ 35kb) 100,000 end sequences

BAC libray 200,000 BAC end sequences


Add into assembly pipeline, data from Transcriptome sequencing:16 runs, a combination of different tissues and conditions for DM and also RH


Acknowledgements

Plant & Food Research is part of the international Potato Genome Sequencing Consortium (PGSC).

For more information, visit http://potatogenome.net.Website going live as of 1st September.

PFR – Lincoln

Jeanne JacobsMark Fiers

Samantha Baldwin

the new zealand institute for plant & food research limited potato genome sequencing consortium,...

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