Step-by-step protocols for rice gamete isolation
A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented.
Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.
KeywordsOryza sativa Egg cell Sperm cell Gametes Transcriptomes
We thank Imtiyaz Khanday, Jonathan Gent, Sarah Anderson and Daniel Jones for helpful advice for optimizing the experimental methods. We thank Debra Skinner for assistance in artwork. This research was funded by the National Science Foundation (Award No. IOS-1547760) and the USDA Agricultural Experiment Station (Project No. CA-D-XXX-6973-H).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Anderson SN, Johnson CS, Jones DS, Conrad LJ, Gou X, Russell SD, Sundaresan V (2013) Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. Plant J 76(5):729–741. https://doi.org/10.1111/tpj.12336 CrossRefGoogle Scholar
- Anderson SN, Johnson CS, Chesnut J, Jones DS, Khanday I, Woodhouse M, Li C, Conrad LJ, Russell SD, Sundaresan V (2017) The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes. Dev Cell 43(3):349.e4–358.e4. https://doi.org/10.1016/j.devcel.2017.10.005 CrossRefGoogle Scholar
- Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Schwartz DC, Tanaka T, Wu J, Zhou S, Childs KL, Davidson RM, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee SS, Kim J, Numa H, Itoh T, Buell CR, Matsumoto T (2013) Improvement of the Oryza sativa nipponbare reference genome using next generation sequence and optical map data. Rice 6(1):3–10. https://doi.org/10.1186/1939-8433-6-4 CrossRefGoogle Scholar
- Ohnishi T, Takanashi H, Mogi M, Takahashi H, Kikuchi S, Yano K, Okamoto T, Fujita M, Kurata N, Tsutsumi N (2011) Distinct gene expression profiles in egg and synergid cells of rice as revealed by cell type-specific microarrays. Plant Physiol 155(2):881–891. https://doi.org/10.1104/pp.110.167502 CrossRefGoogle Scholar
- Russell SD, Gou XP, Wong CE, Wang X, Yuan T, Wei XP, Bhalla PL, Singh MB (2012) Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage. New Phytol 195(3):560–573. https://doi.org/10.1111/j.1469-8137.2012.04199.x CrossRefGoogle Scholar