Sequencing-based large-scale genomics approaches with small numbers of isolated maize meiocytes

Stefanie Dukowic-Schulze,Thiruvarangan Ramaraj,Joann Mudge,Changbin Chen,Anitha Sundararajan

doi:10.3389/fpls.2014.00057

Stefanie Dukowic-Schulze, Thiruvarangan Ramaraj + Show 3 more

Open Access

https://doi.org/10.3389/fpls.2014.00057

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Abstract

High-throughput sequencing has become the large-scale approach of choice to study global gene expression and the distribution of specific chromatin marks and features. However, the limited availability of large amounts of purified cells made it very challenging to apply sequencing-based techniques in plant meiosis research in the past. In this paper, we describe a method to isolate meiocytes from maize anthers and detailed protocols to successfully perform RNA-seq, smRNA-seq, H3K4me3-ChIP-seq, and DNA bisulfite conversion sequencing with 5000–30,000 isolated maize male meiotic cells. These methods can be adjusted for other flowering plant species as well.

Highlights

Plant meiosis research has a long and fruitful history, started by Gregor Mendel and his heredity studies in peas even before DNA was discovered (Mendel, 1865; Kemp, 2002)
The advancements of modern light microscopy facilitated cytological studies of meiosis in many plant species in the last century, and especially cytogenetics done in maize (Rhoades, 1955) propelled the knowledge of meiosis and meiotic recombination forward
We describe here how this capillary collection of meiocytes (CCM) and diverse large-scale downstream analyses can be applied to maize (Figure 1); CCM is not limited to the two model organisms Arabidopsis and maize, and can be adapted for other species, as we did, for example, in rice

Summary

Introduction

Plant meiosis research has a long and fruitful history, started by Gregor Mendel and his heredity studies in peas even before DNA was discovered (Mendel, 1865; Kemp, 2002). The advancements of modern light microscopy facilitated cytological studies of meiosis in many plant species in the last century, and especially cytogenetics done in maize (Rhoades, 1955) propelled the knowledge of meiosis and meiotic recombination forward. In the field of plant meiosis, large-scale transcriptome studies were the first global approaches performed using microarray or sequencing technologies on isolated meiocytes (Chen et al, 2010; Libeau et al, 2011; Yang et al, 2011). Studies began to map and correlate sites of recombination and chromatin features in plants with high resolution (Giraut et al, 2011; Lu et al, 2012; Choi et al, 2013)

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