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Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA

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Haplotyping

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1551))

Abstract

During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp–kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

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References

  1. Osman K, Higgins JD, Sanchez-Moran E et al (2011) Pathways to meiotic recombination in Arabidopsis thaliana. New Phytol 190:523–544

    Article  CAS  PubMed  Google Scholar 

  2. Kauppi L, Jeffreys AJ, Keeney S (2004) Where the crossovers are: recombination distributions in mammals. Nat Rev Genet 5:413–424

    Article  CAS  PubMed  Google Scholar 

  3. Villeneuve AM, Hillers KJ (2001) Whence meiosis? Cell 106:647–650

    Article  CAS  PubMed  Google Scholar 

  4. Barton NH, Charlesworth B (1998) Why sex and recombination? Science 281:1986–1990

    Article  CAS  PubMed  Google Scholar 

  5. Keeney S, Neale MJ (2006) Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation. Biochem Soc Trans 34:523–525

    Article  CAS  PubMed  Google Scholar 

  6. De Massy B (2013) Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes. Annu Rev Genet 47:563–599

    Article  PubMed  Google Scholar 

  7. Mercier R, Mézard C, Jenczewski E et al (2014) The molecular biology of meiosis in plants. Annu Rev Plant Biol. doi:10.1146/annurev-arplant-050213-035923

    PubMed  Google Scholar 

  8. Baudat F, Imai Y, de Massy B (2013) Meiotic recombination in mammals: localization and regulation. Nat Rev Genet 14:794–806

    Article  CAS  PubMed  Google Scholar 

  9. Choi K, Henderson IR (2015) Meiotic recombination hotspots – a comparative view. Plant J 83:52–61

    Article  CAS  PubMed  Google Scholar 

  10. Coop G, Przeworski M (2006) An evolutionary view of human recombination. Nat Rev Genet 8:23–34

    Article  PubMed  Google Scholar 

  11. Yelina NE, Choi K, Chelysheva L et al (2012) Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genet 8, e1002844

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Choi K, Zhao X, Kelly KA et al (2013) Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters. Nat Genet 45:1327–1336

    Article  CAS  PubMed  Google Scholar 

  13. Drouaud J, Khademian H, Giraut L et al (2013) Contrasted patterns of crossover and non-crossover at Arabidopsis thaliana meiotic recombination hotspots. PLoS Genet 9:e1003922

    Article  PubMed  PubMed Central  Google Scholar 

  14. Tiemann-Boege I, Calabrese P, Cochran DM et al (2006) High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing. PLoS Genet 2:e70

    Article  PubMed  PubMed Central  Google Scholar 

  15. Baudat F, de Massy B (2007) Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot. PLoS Genet 3:e100

    Article  PubMed  PubMed Central  Google Scholar 

  16. Cole F, Keeney S, Jasin M (2010) Comprehensive, fine-scale dissection of homologous recombination outcomes at a hot spot in mouse meiosis. Mol Cell 39:700–710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Berg IL, Neumann R, Sarbajna S et al (2011) Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations. Proc Natl Acad Sci U S A 108:12378–12383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kauppi L, May CA, Jeffreys AJ (2009) Analysis of meiotic recombination products from human sperm. Methods Mol Biol. doi:10.1007/978-1-59745-527-5

    PubMed  Google Scholar 

  19. Jeffreys AJ, Kauppi L, Neumann R (2001) Intensely punctate meiotic recombination in the class II region of the major histocompatibility complex. Nat Genet 29:217–222

    Article  CAS  PubMed  Google Scholar 

  20. Arbeithuber B, Betancourt AJ, Ebner T, Tiemann-Boege I (2015) Crossovers are associated with mutation and biased gene conversion at recombination hotspots. Proc Natl Acad Sci U S A 112:2109–2114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. De Boer E, Jasin M, Keeney S (2015) Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice. Genes Dev 29:1721–1733

    Article  PubMed  PubMed Central  Google Scholar 

  22. De Boer E, Jasin M, Keeney S (2013) Analysis of recombinants in female mouse meiosis. Methods Mol Biol 957:19–45

    Article  PubMed  Google Scholar 

  23. Drouaud J, Mézard C (2011) Characterization of meiotic crossovers in pollen from Arabidopsis thaliana. Methods Mol Biol 745:223–249

    Article  CAS  PubMed  Google Scholar 

  24. Yelina NE, Lambing C, Hardcastle TJ et al (2015) DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Genes Dev 29:2183–2202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Khademian H, Giraut L, Drouaud J, Mézard C (2013) Characterization of meiotic non-crossover molecules from Arabidopsis thaliana pollen. Methods Mol Biol 990:177–190

    Article  CAS  PubMed  Google Scholar 

  26. Auton A, McVean G (2012) Estimating recombination rates from genetic variation in humans. Methods Mol Biol 856:217–237

    Article  CAS  PubMed  Google Scholar 

  27. Charlesworth B, Charlesworth D (2010) Elements of evolutionary genetics. Roberts and Company, Englewood, CO

    Google Scholar 

  28. Weigel D, Nordborg M (2015) Population genomics for understanding adaptation in wild plant species. Annu Rev Genet 49:315–338

    Article  CAS  PubMed  Google Scholar 

  29. Auton A, McVean G (2007) Recombination rate estimation in the presence of hotspots. Genome Res 17:1219–1227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fearnhead P (2006) SequenceLDhot: detecting recombination hotspots. Bioinformatics 22:3061–3066

    Article  CAS  PubMed  Google Scholar 

  31. Drouaud J, Mercier R, Chelysheva L et al (2007) Sex-specific crossover distributions and variations in interference level along Arabidopsis thaliana chromosome 4. PLoS Genet 3:12

    Article  Google Scholar 

  32. Giraut L, Falque M, Drouaud J et al (2011) Genome-wide crossover distribution in Arabidopsis thaliana meiosis reveals sex-specific patterns along chromosomes. PLoS Genet 7:e1002354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Salomé PA, Bomblies K, Fitz J et al (2012) The recombination landscape in Arabidopsis thaliana F2 populations. Heredity (Edinb) 108:447–455

    Article  Google Scholar 

  34. Wijnker E, Velikkakam James G, Ding J et al (2013) The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana. Elife 2, e01426

    Article  PubMed  PubMed Central  Google Scholar 

  35. Shilo S, Melamed-Bessudo C, Dorone Y et al (2015) DNA crossover motifs associated with epigenetic modifications delineate open chromatin regions in Arabidopsis. Plant Cell 27:tpc.15.00391. doi:10.1105/tpc.15.00391

    Article  Google Scholar 

  36. Hellsten U, Wright KM, Jenkins J et al (2013) Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing. Proc Natl Acad Sci U S A 110:19478–19482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dooner HK (1986) Genetic fine structure of the BRONZE Locus in maize. Genetics 113:1021–1036

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Brown J, Sundaresan V (1991) A recombination hotspot in the maize A1 intragenic region. Theor Appl Genet 81:185–188

    Article  CAS  PubMed  Google Scholar 

  39. Saintenac C, Faure S, Remay A et al (2011) Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot. Chromosoma 120:185–198

    Article  CAS  PubMed  Google Scholar 

  40. Chodavarapu RK, Feng S, Bernatavichute YV et al (2010) Relationship between nucleosome positioning and DNA methylation. Nature 466:388–392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zilberman D, Coleman-Derr D, Ballinger T, Henikoff S (2008) Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456:125–129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhang X, Bernatavichute YV, Cokus S et al (2009) Genome-wide analysis of mono-, di- and trimethylation of histone H3 lysine 4 in Arabidopsis thaliana. Genome Biol 10:R62

    Article  PubMed  PubMed Central  Google Scholar 

  43. Liu S, Yeh C-T, Ji T et al (2009) Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome. PLoS Genet 5:13

    CAS  Google Scholar 

  44. Copenhaver GP, Nickel K, Kuromori T et al (1999) Genetic definition and sequence analysis of Arabidopsis centromeres. Science 286:2468–2474

    Article  CAS  PubMed  Google Scholar 

  45. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  46. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Li H, Handsaker B, Wysoker A et al (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  50. R Development Core Team (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK

    Kyuha Choi, Nataliya E. Yelina, Heïdi Serra & Ian R. Henderson

Authors
  1. Kyuha Choi
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  2. Nataliya E. Yelina
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  3. Heïdi Serra
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  4. Ian R. Henderson
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Corresponding author

Correspondence to Ian R. Henderson .

Editor information

Editors and Affiliations

  1. Johannes Kepler University Institute of Biophysics, Linz, Austria

    Irene Tiemann-Boege

  2. Vetmeduni Vienna InstitutPopulationsgenetik, Wien, Austria

    Andrea Betancourt

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Choi, K., Yelina, N.E., Serra, H., Henderson, I.R. (2017). Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA. In: Tiemann-Boege, I., Betancourt, A. (eds) Haplotyping. Methods in Molecular Biology, vol 1551. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6750-6_2

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  • DOI: https://doi.org/10.1007/978-1-4939-6750-6_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6748-3

  • Online ISBN: 978-1-4939-6750-6

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