Advertisement

Plant Meiosis pp 281-300 | Cite as

Quantification of Recombination Rate and Segregation Distortion by Genotyping and Sequencing of Single Pollen Nuclei

  • Steven Dreissig
  • Jörg Fuchs
  • Axel Himmelbach
  • Martin MascherEmail author
  • Andreas HoubenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)

Abstract

Meiosis is a specialized cell division during which homologous chromosomes can exchange genetic material through recombination. This mechanism generates novel allelic combinations, which can be exploited by plant breeders to achieve crop improvement. Pollen grains are the haploid products of meiosis required in fertilization. Here, we describe two approaches to measure meiotic recombination in single haploid pollen nuclei. Pollen nuclei are first separated by fluorescence-activated cell-sorting. Afterwards, the DNA of single pollen nuclei can be amplified by multiple-displacement-amplification using Phi29 DNA polymerase and meiotic recombination events can be measured using KASP markers. Alternatively, the PicoPLEX DNA-seq kit can be used to amplify the DNA of single pollen nuclei followed by library preparation for whole-genome sequencing and subsequent bioinformatic analysis.

Key words

Pollen Single cell Recombination Segregation distortion Flow sorting Whole-genome amplification 

Notes

Acknowledgments

We thankfully acknowledge Petr Cápal for his advice on single-cell whole-genome-amplification. Furthermore, we like to acknowledge Nils Stein (IPK, Gatersleben) for providing “Morex” × “Barke” F1 seeds and Sandra Dreisslein as well as Ines Walde (IPK, Gatersleben) for their excellent technical assistance. This work was supported by IPK Gatersleben.

References

  1. 1.
    Villeneuve AM, Hillers KJ (2001) Whence meiosis? Cell 106:647–650CrossRefGoogle Scholar
  2. 2.
    Stapley J, Feulner PGD, Johnston SE et al (2017) Variation in recombination frequency and distribution across eukaryotes: patterns and processes. Philos Trans R Soc Lond Ser B Biol Sci 372:20160455CrossRefGoogle Scholar
  3. 3.
    Ramsay L, Colas I, Waugh R (2014) Modulation of meiotic recombination. In: Kumlehn J, Stein N (eds) Biotechnological approaches to barley improvement. Springer, Berlin Heidelberg, pp 311–329CrossRefGoogle Scholar
  4. 4.
    Choi K (2017) Advances towards controlling meiotic recombination for plant breeding. Mol Cells 40:814–822CrossRefGoogle Scholar
  5. 5.
    Wang J, Fan HC, Behr B et al (2012) Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 150:402–412CrossRefGoogle Scholar
  6. 6.
    Gawad C, Koh W, Quake SR (2016) Single-cell genome sequencing: current state of the science. Nat Rev Genet 17:175–188CrossRefGoogle Scholar
  7. 7.
    Gole J, Gore A, Richards A et al (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31:1126–1132CrossRefGoogle Scholar
  8. 8.
    Li X, Li L, Yan J (2015) Dissecting meiotic recombination based on tetrad analysis by single-microspore sequencing in maize. Nat Commun 6:6648CrossRefGoogle Scholar
  9. 9.
    Li X, Meng D, Chen S et al (2017) Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction. Nat Commun 8:991CrossRefGoogle Scholar
  10. 10.
    Dreissig S, Fuchs J, Himmelbach A et al (2017) Sequencing of single pollen nuclei reveals meiotic recombination events at megabase resolution and circumvents segregation distortion caused by postmeiotic processes. Front Plant Sci 8:1620CrossRefGoogle Scholar
  11. 11.
    Dreissig S, Fuchs J, Cápal P et al (2015) Measuring meiotic crossovers via multi-locus genotyping of single pollen grains in barley. PLoS One 10:e0137677CrossRefGoogle Scholar
  12. 12.
    Galbraith DW, Harkins KR, Maddox JM et al (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051CrossRefGoogle Scholar
  13. 13.
    Kron P, Husband BC (2012) Using flow cytometry to estimate pollen DNA content: improved methodology and applications. Ann Bot 110:1067–1078CrossRefGoogle Scholar
  14. 14.
    De Storme N, Geelen D (2011) The Arabidopsis mutant jason produces unreduced first division restitution male gametes through a parallel/fused spindle mechanism in meiosis II. Plant Physiol 155:1403–1415CrossRefGoogle Scholar
  15. 15.
    Himmelbach A, Knauft M, Stein N (2014) Plant sequence capture optimised for Illumina sequencing. Bio-protocol 4:e1166CrossRefGoogle Scholar
  16. 16.
    Mascher M, Muehlbauer GJ, Rokhsar DS et al (2013) Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ). Plant J 76:718–727CrossRefGoogle Scholar
  17. 17.
    Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetjournal 17:10–12Google Scholar
  18. 18.
    Mascher M, Gundlach H, Himmelbach A et al (2017) A chromosome conformation capture ordered sequence of the barley genome. Nature 544:427–433CrossRefGoogle Scholar
  19. 19.
    Aliyeva-Schnorr L, Beier S, Karafiátová M et al (2015) Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H. Plant J 84:385–394CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)Stadt SeelandGermany

Personalised recommendations