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Use of RAD Sequencing to Create a Meiotic Recombination Map in Arabidopsis

Use of RAD Sequencing to Create a Meiotic Recombination Map in Arabidopsis. Natasha Elina Plant Sciences, University of Cambridge Edinburgh October 21 st 2009. Homologous chromosomes. Chiasma. Recombinant chromatids. Meiotic Recombination.

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Use of RAD Sequencing to Create a Meiotic Recombination Map in Arabidopsis

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  1. Use of RAD Sequencing to Create a Meiotic Recombination Map in Arabidopsis Natasha Elina Plant Sciences, University of Cambridge Edinburgh October 21st 2009

  2. Homologous chromosomes Chiasma Recombinant chromatids Meiotic Recombination

  3. Recombination is not evenly distributed along the chromosome – ‘hot’ and ‘cold’ spots Drouaud et al., Genome Research,2006

  4. What is the distribution of cross-over frequencies (a fine map)? What genomic features do cross-over distributions correlate with?

  5. Why do we want to know that? Plant breeding, population genetics

  6. Model Plant – Arabidopsis thaliana Genome completely sequenced Sequence information available for different ecotypes Epigenomic information (siRNA database, DNA methylation, etc) Mutants that change this information More cross-over in the male than in female meioses; sex average is 8.9

  7. High Resolution Mapping of Meiotic Recombination Points Columbia-0 F1 Landsberg erecta Back-crossing F1 to one of the parents X Columbia-0 RAD sequencing this population and making a recombinant map

  8. Two Arabidopsis Ecotypes Columbia-0 and Landsberg erecta 1% sequence divergence ~ 120,000 total polymorphisms Col-0 Ler

  9. Statistical Rationale for the Project Arabidopsis genome – 5 chromosomes Whole genome ~125Mb Average of 8.9 cross-overs per meiosis ~240 SNPs/chromosome; 1200 SNPs total 200 kb ~ 1 cM 1200 SNP markers in 500 F2 plants will give a 95% likelihood of observing an average of 10 CO events per interval Looking at the same SNPs in all plants: RAD mapping (Baird et al., PLoS ONE 2008) Krys Kelly

  10. Distribution of SNPs linked to the restriction enzyme Krys Kelly

  11. RAD Mapping DNA digestion Library amplification Sonication Size selection 1 – sample 2- DNA ladder RAD library profile

  12. Adapters and Bar Coding genomic DNA P1 adapter SNP SNP SNP P2 adapter Illumina sequence read length – 35-100 nt Based on 31x depth with Illumina, we need 0.5 mln reads per plant P1 adapter: Contains a barcode: four base code followed by the fifth ‘checksum’ base; Barcode sequences – a combination of most divergent ones Bar-codes used: A - cgtga, B – gtcga, C – agcgc, D - tatga P1 and P2 adapters: 5’ phosphorylated 3’ base is linked through a phosphorothioate bond

  13. Work in progress (pilot study) Expected: Col-0, homozygous Col-0 Ler, homozygous Ler Col, Ler heterozygous F1:ColxLer F2 Col, Ler, recombination point(s) Total number of reads: 8,491,734 After de-multiplexing:7,936,046 Percentage recovered: 93%

  14. Workflow – from DNA to a Cross-Over Frequency Map RAD library population of recombinants Illumina sequencing data Aligning sequences to the reference genome genotype calling Cross-over frequency map

  15. Future Plans DNA methylation profile Expression profile in met1 Cross-over frequencies Drouaud et al., Genome Research,2006; Zhang et al., Cell 2006; Zilberman et al., Nature Genetics 2007; Cokus et al., Nature 2008

  16. Acknowledgements Paul Etter Eric Johnson Krys Kelly Kim Rutheford Ian Henderson Liz Alvey David Baulcombe CRUK Cancer Research Institute James Hadfield Nik Mattews Kevin Howe Rory Stark

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