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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Villeneuve AM, Hillers KJ (2001) Whence meiosis? Cell 106:647–650
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 372:20160455
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 311–329
Choi K (2017) Advances towards controlling meiotic recombination for plant breeding. Mol Cells 40:814–822
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 150:402–412
Gawad C, Koh W, Quake SR (2016) Single-cell genome sequencing: current state of the science. Nat Rev Genet 17:175–188
Gole J, Gore A, Richards A et al (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31:1126–1132
Li X, Li L, Yan J (2015) Dissecting meiotic recombination based on tetrad analysis by single-microspore sequencing in maize. Nat Commun 6:6648
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 8:991
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 8:1620
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:e0137677
Galbraith DW, Harkins KR, Maddox JM et al (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051
Kron P, Husband BC (2012) Using flow cytometry to estimate pollen DNA content: improved methodology and applications. Ann Bot 110:1067–1078
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 155:1403–1415
Himmelbach A, Knauft M, Stein N (2014) Plant sequence capture optimised for Illumina sequencing. Bio-protocol 4:e1166
Mascher M, Muehlbauer GJ, Rokhsar DS et al (2013) Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ). Plant J 76:718–727
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetjournal 17:10–12
Mascher M, Gundlach H, Himmelbach A et al (2017) A chromosome conformation capture ordered sequence of the barley genome. Nature 544:427–433
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 84:385–394
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.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dreissig, S., Fuchs, J., Himmelbach, A., Mascher, M., Houben, A. (2020). Quantification of Recombination Rate and Segregation Distortion by Genotyping and Sequencing of Single Pollen Nuclei. In: Pradillo, M., Heckmann, S. (eds) Plant Meiosis. Methods in Molecular Biology, vol 2061. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9818-0_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9818-0_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9817-3
Online ISBN: 978-1-4939-9818-0
eBook Packages: Springer Protocols