Protocol for Chromatin Immunoprecipitation of Meiotic‐Stage‐Specific Tomato Anthers

Abstract

Interactions occurring between DNA and proteins across the nuclear genome regulate numerous processes, including meiosis. Meiosis ensures genetic variation and balanced segregation of homologous chromosomes. It involves complex DNA-protein interactions across the entire genome to regulate a broad range of processes, including formation and repair of double-strand DNA breaks (DSBs), chromosome compaction, homolog pairing, synapsis, and homologous recombination. The latter meiotic event, meiotic recombination, often occurs at discrete locations in a genome, within a tight time window. The identification of genomic binding sites of meiotic proteins is a major step toward understanding the molecular mechanisms underlying meiotic recombination and provides important information for plant breeding. Collecting meiotic cells from plants is challenging, tedious, and time consuming, since the meiocyte-producing organs, the anthers, are generally small and limited to certain developmental stages of plants. Here we provide a protocol to isolate meiotic-stage-specific anthers and perform ChIP on this material. We have developed a ChIP protocol specifically suited to (1) small amounts of input material and (2) proteins that bind transiently to chromatin and at very low frequency. © 2018 by John Wiley & Sons, Inc.

Technology-driven approaches for meiosis research in tomato and wild relatives

“…A recent study on historical recombination in cultivated and wild tomato shows that DNA transposons including hAT-Tip100 and Stowaway elements show enrichment in hot spots, whereas most class I retrotransposons including the Copia and Gypsy element classes are overrepresented in cold spots of recombination (Fuentes et al 2022 ). Furthermore, hot and cold spots of recombination have been found to respectively associate with high and low chromatin accessibility in tomato (Chouaref 2021 ). These observations suggest an interplay between chromatin accessibility and meiotic recombination that we are just beginning to understand.…”

A computational study of genomic rearrangements in plants

“…The location and distance of these nodules on the chromatin can be measured in micrometers and evaluated per unit length of spread chromatin (Anderson and Stack, 2005). A second and more precise analysis method, replacing the visualization technique, is based on chromatin immunoprecipitation sequencing (ChIP-Seq, Chouaref et al, 2018) or immunofluorescent in situ hybridization (FISH, Sepsi et al, 2018) of recombination-specific proteins bound to chromosomes. By using specific antibodies, the protein complexes together with the DNA that they bind are extracted and subsequently sequenced.…”