Metaphase Chromosome Preparation from Soybean (Glycine max) Root Tips

Abstract

This unit presents a highly reliable protocol to produce and screen metaphase chromosome spreads from root tip cell suspensions of soybean (Glycine max), or other legumes. The procedures represent soybean‐optimized versions of protocols developed for maize. The use of pressurized nitrous oxide to reliably generate metaphase‐arrested chromosomes is crucial to overcoming one of the challenges of working with tiny and numerous soybean chromosomes. © 2017 by John Wiley & Sons, Inc.

Fluorescence In Situ Hybridization for Glycine max Metaphase Chromosomes

“…This FISH procedure uses chromosome spreads prepared as described in Findley et al (2017). Prior to starting the hybridization procedures, fluorescent DNA FISH probes must be synthesized and purified (nick translation probes) or ordered from a synthesis facility and diluted into working solutions (fluorochrome-tagged oligonucleotides).…”

“…Twenty previously prepared Glycine max root-tip metaphase chromosome slides prepared as described in Findley et al (2017) are heat treated to denature chromosomes, then subjected to a blocking step that incorporates a commercially prepared sonicated salmon sperm DNA solution (sssDNA). If individual slides must be tracked, they can be marked with a diamond-tip pencil, which is more reliable than ink and most slide labels.…”

“…Probes made from larger DNA fragments (e.g., 100-kb BAC inserts of single-copy genes/regions) are proportionally brighter, and thus more desirable. Newer technologies, (Beliveau et al, 2015;Fominaya, Loarce, González, & Ferrer, 2016) may permit identification of targets smaller than 10 kb, such as single transgenes, and would likely be compatible with the metaphase soybean chromosome spread slides prepared in Findley et al (2017). In contrast, much smaller dsDNA fragments (e.g., 400 bp for rDNA) or even oligonucleotides can be used as FISH probes if the probe target is present in highcopy tandem arrays (e.g., the SB86 or CentGm repeats; Findley et al, 2010).…”

“…It is important to emphasize that the quality of images obtainable with these FISH protocols depends foremost on the use of high‐quality chromosome spreads (see Findley et al., ). Once good spreads are obtained, the next step is to verify that the hybridization reaction is working well.…”

“…In this article, several fluorescence in situ hybridization (FISH) protocols are outlined for metaphase soybean chromosome spreads prepared as described in Findley, Birchler, & Stacey (2017). FISH technology offers a means to rapidly answer a broad range of genome-scale questions (see Commentary).…”